Recombinant constructs and their use in reducing gene expression

ABSTRACT

Recombinant constructs useful for reducing the expression of endogenous mRNA and any substantially similar endogenous mRNA are disclosed. In particular, a recombinant construct comprising, inter alia, a suitable nucleic acid sequence and its reverse complement can be used to alter the expression of any homologous, endogenous RNA (i.e., the target RNA) which is in proximity to this suitable nucleic acid sequence.

FIELD OF THE INVENTION

This invention relates to reducing gene expression and, in particular,to recombinant constructs useful for reducing the expression ofendogenous mRNA and any substantially similar endogenous mRNA.

BACKGROUND OF THE INVENTION

Plant development is a complex physiological and biochemical processrequiring the coordinated expression of many genes. The production ofnew plant varieties with improved nutritional or disease-resistanttraits can be achieved by modifying this coordinated pattern of geneexpression. Recombinant DNA techniques have made it possible to alterthe expression patterns of individual, specific plant genes withoutdirectly affecting the expression of other plant genes. In this way, theexpression pattern of an individual gene can be either enhanced ordiminished in the whole plant, in specific tissues, or in developmentalstages. Thus, it is now routine to construct transgenes with definedpromoters and terminators and express them in a variety of organisms.

However, there are some reports in the literature that some introducedtransgenes do not have the expected expression patterns. Theseunexpected expression patterns are seen in organisms as diverse asnematodes and plants. For example, some plants receiving transgeniccopies of an endogenous gene under the control of a strong promoter,sometimes fail to accumulate mRNA for that gene. Furthermore, all mRNAfrom endogenous genes having sequence homology to the transgene alsofail to accumulate mRNA, effectively eliminating the expression of theendogenous gene product. This was discovered originally when chalconesynthase transgenes in petunia caused suppression of the endogenouschalcone synthase genes (Napoli et al (1990) Plant Cell 2:279-289).

The phenomenon was referred to as “cosuppression” since expression ofboth the endogenous gene and the introduced transgene were suppressed(for reviews see Vaucheret et al (1998) Plant J 16:651-659; and Gura(2000) Nature 404:804-808). Cosuppression technology constitutes thesubject matter of U.S. Pat. No. 5,231,020 which issued to Jorgensen etal on Jul. 27, 1999. Cosuppression is also referred to as “genesilencing” or post-transcriptional gene silencing (PTGS) by plantbiologists, “RNA interference” by those studying worms and flies(Montgomery and Fire (1998) TIG 14:255-258; Fire et al (1998) Nature391:806-811; Hammond et al (2000) Nature 404:293-296; and PCTApplication No. WO 99/32619 published Jul. 1, 1999), and “quelling” byresearchers working with fungi (Romano and Macino (1992) Mol Microbiol6:3343-3353).

The mechanisms by which the expression of a specific gene is inhibitedby either antisense or sense RNA genes are not clearly understood andthe frequencies of obtaining the desired down regulation in a transgenicplant are generally low and vary with the gene, the strength of itspromoter and specificity, the method of transformation, and thecomplexity of transgene insertion events. (Grant (1999) Cell 96:303-306;and Selker (1999) Cell 97:157-160.)

The speculation is that PTGS is an ancient self-defense mechanismevolved to combat infection by viruses and transposons. It appears thatthis pathogen-derived resistance is triggered by the presence in thehost's cells of double-stranded RNA (dsRNA) or some other aberrantnucleic acid, which are indicative of a viral assault. Normally, the RNAmoving freely around a cell should be single-stranded messenger RNA(mRNA) which is the intermediate between host genes and the proteinsthey encode. When the aberrant RNA invades then any mRNAs matching theinvading nucleic acid's sequence are shut down. If the trigger ishomologous to part of the host's genetic sequence, then both the hostand viral genes are silenced (Baulcombe (1996) Plant Cell 8:1833-1844).WO 99/15682 which published on Apr. 1, 1999 and WO 98/36083 whichpublished on Aug. 20, 1998 describe gene silencing materials andmethods. These publications describe, inter alia, the silencing of plantgenomic gene expression by introducing expression constructs containingplant viral nucleic acid sequences coupled to whole, or partial, genesequences homologous to the target genes to be silenced.

WO 99/53050, which published on Oct. 21, 1999, describes chimericconstructs encoding RNA molecules directed towards a target nucleic acidwhich are comprised of sense and antisense sequences, such that theexpressed RNA is capable of forming an intramolecular double-strandedRNA structure. The expression of these RNA in transgenic organismsresults in gene silencing of the all homologous target nucleic acidsequences within the cell.

U.S. Pat. No. 5,942,657, issued to Bird et al on Aug. 25, 1999, and WO93/23551, which published on Nov. 25, 1993, describe coordinatedinhibition of plant gene expression in which two or more genes areinhibited by introducing a single control gene having distinct DNAregions homologous to each of the target genes and a promoter operablein plants adapted to transcribe from such distinct regions RNA thatinhibits expression of each of the target genes.

The present invention describes the use of suitable DNA sequences or RNAsequences derived therefrom, as is discussed below, in ways whichhere-to-fore have not been previously described. These sequences, andtheir reverse complements, can be used to reduce the expression of anyendogenous genomic sequence that shares substantial similarity tonucleic acid fragment which is in proximity to the DNA sequence or RNAsequence derived therefrom. The details of this phenomenon are describedherein.

SUMMARY OF THE INVENTION

This invention concerns a recombinant construct comprising a promoteroperably linked to a DNA sequence which, when expressed by a hostproduces an RNA having:

(a) homology to at least one target mRNA expressed by the host,

(b) two complementary RNA regions which are unrelated to any endogenousRNA in the host, and which are in proximity to (a),

wherein the expressed RNA reduces the expression of the target mRNA orany substantially similar endogenous mRNA.

In a second embodiment, this invention concerns a recombinant constructcomprising a promoter operably linked to a DNA sequence which, whenexpressed by a host, produces an RNA having:

(a) homology to at least one target mRNA expressed by the host,

(b) an RNA region unrelated to any endogenous RNA in the host andlocated 5′ to (a), and

(c) the reverse complement of the RNA in (b) located 3′ to (a),

wherein the expressed RNA reduces the expression of the target mRNA orany substantially similar endogenous mRNA.

In a third embodiment, this invention concerns a recombinant constructcomprising a promoter operably linked to a DNA sequence which, whenexpressed by a host, produces an RNA having:

(a) homology to at least one target mRNA expressed by the host, and

(b) two complementary RNA regions which are unrelated to any endogenousRNA in the host, and which are located 5′ to (a),

wherein the expressed RNA reduces the expression of the target mRNA orany substantially similar endogenous mRNA.

In a fourth embodiment, this invention concerns a recombinant constructcomprising a promoter operably linked to a DNA sequence which, whenexpressed by a host, produces an RNA having:

(a) homology to at least one target mRNA expressed by the host, and

(b) two complementary RNA regions which are unrelated to any endogenousRNA in the host, and which are located 3′ to (a),

wherein the expressed RNA reduces the expression of the target mRNA orany substantially similar endogenous mRNA.

In a fifth embodiment, this invention concerns a recombinant constructcomprising a promoter operably linked to a DNA sequence which, whenexpressed by a host, produces an RNA having:

(a) homology to at least one target mRNA expressed by the host, and

(b) two complementary RNA regions which are unrelated to any endogenousRNA in the host, and which are located within (a),

wherein the expressed RNA reduces the expression of the target mRNA orany substantially similar endogenous mRNA.

In another aspect of any of the foregoing recombinant constructs, theRNA region or regions which are unrelated to any endogenous RNA in thehost comprise a synthetic, non-naturally occurring RNA sequence.

In still another aspect of any of the foregoing recombinant constructs,the RNA region or regions which are unrelated to any endogenous RNA inthe host do not comprise plant viral RNA.

In a sixth embodiment, this invention concerns a method for reducingexpression of a target mRNA or any substantially similar endogenous mRNAwhich comprises:

(a) transforming a host with any of the above-described recombinantconstructs; and

(b) selecting hosts which have reduced expression of the target mRNA orany substantially similar endogenous mRNA.

In a seventh, embodiment, this invention concerns a recombinantconstruct comprising an RNA having:

(a) homology to at least one target mRNA expressed by a host,

(b) two complementary RNA regions which are unrelated to any endogenousRNA in the host, and which are in proximity to (a),

wherein the RNA, when introduced into the host, reduces the expressionof the target mRNA or any substantially similar endogenous mRNA.

In an eighth embodiment, this invention concerns a recombinant constructcomprising an RNA having:

(a) homology to at least one target mRNA expressed by a host,

(b) an RNA region unrelated to any endogenous RNA in the host andlocated 5′ to (a), and

(c) the reverse complement of the RNA in (b) located 3′ to (a),

wherein the RNA, when introduced into the host, reduces the expressionof the target mRNA or any substantially similar endogenous mRNA.

In a ninth embodiment, this invention concerns a recombinant constructcomprising an RNA having:

(a) homology to at least one target mRNA expressed by the host, and

(b) two complementary RNA regions which are unrelated to any endogenousRNA in the host, and which are located 5′ to (a),

wherein the RNA, when introduced into the host, reduces the expressionof the target mRNA or any substantially similar endogenous mRNA.

In a tenth embodiment, this invention concerns a recombinant constructcomprising an RNA having:

(a) homology to at least one target mRNA expressed by the host, and

(b) two complementary RNA regions which are unrelated to any endogenousRNA in the host, and which are located 3′ to (a),

wherein the RNA, when introduced into the host, reduces the expressionof the target mRNA or any substantially similar endogenous mRNA.

In an eleventh embodiment, this invention concerns a recombinantconstruct comprising an RNA having:

(a) homology to at least one target mRNA expressed by the host, and

(b) two complementary RNA regions which are unrelated to any endogenousRNA in the host, and which are located within (a),

wherein the RNA, when introduced into the host, reduces the expressionof the target mRNA or any substantially similar endogenous mRNA.

In another aspect of any of the foregoing RNAs, the RNA region orregions which are unrelated to any endogenous RNA in the host comprise asynthetic, non-naturally occurring RNA sequence.

In still another aspect of any of the foregoing recombinant constructs,the RNA region or regions which are unrelated to any endogenous RNA inthe host do not comprise plant viral RNA.

In a twelfth embodiment, this invention concerns a method for reducingexpression of a target mRNA or any substantially similar endogenous mRNAwhich comprises:

(a) introducing into a host any of the above-described RNAs; and

(b) selecting hosts which have reduced expression of the target mRNA orany substantially similar endogenous mRNA.

In a thirteenth embodiment this invention concerns, a recombinantconstruct comprising a promoter operably linked to a DNA sequence which,when expressed by a host produces an RNA having:

(a) homology to at least one target mRNA expressed by the host,

(b) two complementary RNA regions which are encoded by any nucleic acidsequence in the genome of the host provided that said sequence does notencode the target mRNA or any sequence that is substantially similar tothe target mRNA and said regions are in proximity to (a),

wherein the expressed RNA reduces the expression of the target mRNA orany substantially similar endogenous mRNA.

In a fourteenth embodiment this invention concerns, a recombinantconstruct comprising a promoter operably linked to a DNA sequence which,when expressed by a host produces an RNA having:

(a) homology to at least one target mRNA expressed by the host,

(b) an RNA region encoded by any nucleic acid sequence in the genome ofthe host provided that said sequence does not encode the target mRNA orany sequence that is substantially similar to the target mRNA andlocated 5′ to (a), and

(c) the reverse complement of the nucleic acid in (b) located 3′ to (a),

wherein the expressed RNA reduces the expression of the target mRNA orany substantially similar endogenous mRNA.

In a fifteenth embodiment this invention concerns, a recombinantconstruct comprising a promoter operably linked to a DNA sequence which,when expressed by a host produces an RNA having:

(a) homology to at least one target mRNA expressed by the host, and

(b) two complementary RNA regions which are encoded by any nucleic acidsequence in the genome of the host provided that said sequence does notencode the target mRNA or any sequence that is substantially similar tothe target mRNA, and which regions are located 5′ to (a),

wherein the expressed RNA reduces the expression of the target mRNA orany substantially similar endogenous mRNA.

In a sixteenth embodiment this invention concerns, a recombinantconstruct comprising a promoter operably linked to a DNA sequence which,when expressed by a host produces an RNA having:

(a) homology to at least one target mRNA expressed by the host, and

(b) two complementary RNA regions which are encoded by any nucleic acidsequence in the genome of the host provided that said sequence does notencode the target mRNA or any sequence that is substantially similar tothe target mRNA, and which regions are located 3′ to (a),

wherein the expressed RNA reduces the expression of the target mRNA orany substantially similar endogenous mRNA.

In a seventeenth embodiment this invention concerns, a recombinantconstruct comprising a promoter operably linked to a DNA sequence which,when expressed by a host produces an RNA having:

(a) homology to at least one target mRNA expressed by the host, and

(b) two complementary RNA regions which are encoded by any nucleic acidsequence in the genome of the host provided that said sequence does notencode the target mRNA or any sequence that is substantially similar tothe target mRNA, and which regions are located within (a),

wherein the expressed RNA reduces the expression of the target mRNA orany substantially similar endogenous mRNA.

In another aspect of any of the foregoing recombinant constructs, theRNA region or regions which are unrelated to any endogenous RNA in thehost comprise a synthetic, non-naturally occurring RNA sequence.

In still another aspect of any of the foregoing recombinant constructs,the RNA region or regions which are unrelated to any endogenous RNA inthe host do not comprise plant viral RNA.

In an eighteenth embodiment this invention concerns, a method forreducing expression of a target mRNA or any substantially similarendogenous mRNA which comprises:

(a) transforming a host with any of the above-described recombinantconstructs; and

(b) selecting hosts which have reduced expression of the target mRNA orany substantially similar endogenous mRNA.

In a nineteenth embodiment this invention concerns an RNA comprising:

(a) homology to at least one target mRNA expressed by a host,

(b) two complementary RNA regions which are encoded by any nucleic acidsequence in the genome of the host provided that said sequence does notencode the target mRNA or any sequence that is substantially similar tothe target mRNA and which regions are in proximity to (a),

wherein the RNA, when introduced into the host, reduces the expressionof the target mRNA or any substantially similar endogenous mRNA.

In a twentieth embodiment this invention concerns an RNA comprising:

(a) homology to at least one target mRNA expressed by a host,

(b) an RNA region encoded by any nucleic acid sequence in the genome ofthe host provided that said sequence does not encode the target mRNA orany sequence that is substantially similar to the target mRNA and islocated 5′ to (a), and

(c) the reverse complement of the RNA in (b) located 3′ to (a),

wherein the RNA, when introduced into the host, reduces the expressionof the target mRNA or any substantially similar endogenous mRNA.

In a twenty-first embodiment this invention concerns an RNA comprising:

(a) homology to at least one target mRNA expressed by the host, and

(b) two complementary RNA regions which are encoded by any nucleic acidsequence in the genome of the host provided that said sequence does notencode the target mRNA or any sequence that is substantially similar tothe target mRNA and which regions are located 5′ to (a),

wherein the RNA, when introduced into the host, reduces the expressionof the target mRNA or any substantially similar endogenous mRNA.

In a twenty-second embodiment this invention concerns an RNA comprising:

(a) homology to at least one target mRNA expressed by the host, and

(b) two complementary RNA regions which are encoded by any nucleic acidsequence in the genome of the host provided that said sequence does notencode the target mRNA or any sequence that is substantially similar tothe target mRNA, and which regions are located 3′ to (a),

wherein the RNA, when introduced into the host, reduces the expressionof the target mRNA or any substantially similar endogenous mRNA.

In a twenty-third embodiment this invention concerns an RNA comprising:

(a) homology to at least one target mRNA expressed by the host, and

(b) two complementary RNA regions which are encoded by any nucleic acidsequence in the genome of the host provided that said sequence does notencode the target mRNA or any sequence that is substantially similar tothe target mRNA, and which are located within (a),

wherein the RNA, when introduced into the host, reduces the expressionof the target mRNA or any substantially similar endogenous mRNA.

In another aspect of any of the foregoing RNAs, the RNA region orregions which are unrelated to any endogenous RNA in the host comprise asynthetic, non-naturally occurring RNA sequence.

In still another aspect of any of the foregoing recombinant constructs,the RNA region or regions which are unrelated to any endogenous RNA inthe host do not comprise plant viral RNA.

In a twenty-fourth embodiment this invention concerns a method forreducing expression of a target mRNA or any substantially similarendogenous mRNA which comprises:

(a) introducing into a host any of the above-described RNAs; and

(b) selecting hosts which have reduced expression of the target mRNA orany substantially similar endogenous mRNA.

In a twenty-fifth embodiment, this invention concerns a method foridentifying or screening an essential plant gene which comprises:

(a) transforming a plant cell with a recombinant construct comprising aconstitutive promoter wherein said construct is capable of reducingexpression of an essential plant gene with a high degree of frequency;

(b) quantifying all transformed plant cells from step (a);

(c) quantifying all transformed plant cells from a control which doesnot reduce expression of an essential plant gene; and

(d) comparing the quantification of transformed plant cells selectedfrom step (b) with the quantification of transformed plants cellsselected from step (c) wherein the quantification of transformed plantscells selected from step (c) should substantially exceed thequantification of transformed plant cells selected from step (b).

In a twenty-sixth embodiment, this invention concerns a method foridentifying or screening an essential plant gene which comprises:

(a) transforming a plant cell with any of the recombinant constructs ofthe invention comprising a promoter operably linked to a DNA sequenceand which further comprises a constitutive promoter which is capable ofreducing expression of an essential plant gene with a high degree offrequency;

(b) quantifying all transformed plant cells from step (a);

(c) quantifying all transformed plant cells from a control which doesnot reduce expression of an essential plant gene; and

(d) comparing the quantification of transformed plant cells selectedfrom step (b) with the quantification of transformed plants cellsselected from step (c) wherein the quantification of transformed plantscells selected from step (c) should substantially exceed thequantification of transformed plant cells selected from step (b).

BRIEF DESCRIPTION OF THE DRAWING AND SEQUENCE LISTINGS

The invention can be more fully understood from the following detaileddescription and the accompanying drawings and Sequence Listing whichform a part of this application.

FIG. 1 depicts the results of chimerism in experiments on antisense,“classical co-suppression”, and complementary region reduction ofexpression for the soybean gene Fad2, a fatty acid desaturase. Chimerismis a measure of the percentage of individuals isolated in individualtransformed lines that exhibit the phenotype characteristic of thedesired trait.

FIG. 2 shows total soybean sugars visualized after TLC separation. Theraffinose and stachyose sugars are the lowest band in each lane. The“Low 4” lane is isolated from a soybean line known to have very lowlevels of raffinose/stachyose sugars. The two “GAS-EL” lines both havelower levels of raffinose/stachyose than are found in the surroundinglines indicating that the GAS1/GAS2 fragments contained within the ELconstruct are suppressing galactinol synthase activity in these lines.

The attached Sequence Listing (SEQ ID NOs:1-35) describe oligonucleotidesequences used in the design of various plasmids described herein, orthe sequence of the complementary regions found in some of the plasmids.

SEQ ID NO:1 is the sequence of an oligonucleotide primer used in apolymerase chain reaction (PCR) amplification of the soybean Fad2-1 genefor insertion into plasmid pKS67 to produce plasmid pKS91.

SEQ ID NO:2 is the sequence of an oligonucleotide primer used in a PCRamplification of the soybean Fad2-1 gene for insertion into plasmidpKS67 to produce plasmid pKS91.

SEQ ID NO:3 is the sequence of an oligonucleotide primer used in a PCRamplification of the soybean Fad2-1 gene for insertion into plasmidpKS67 to produce plasmid pKS91.

SEQ ID NO:4 is the sequence of an oligonucleotide primer used in a PCRamplification of the soybean Fad2-1 gene for insertion into plasmidpKS67 to produce plasmid pKS91.

SEQ ID NO:5 is the sequence of an oligonucleotide primer used in a PCRamplification of the soybean Fad2-1 gene for insertion into plasmidpKS67 to produce plasmid pKS91.

SEQ ID NO:6 is the sequence of an oligonucleotide primer used in a PCRamplification of the soybean Fad2-1 gene for insertion into plasmidpKS67 to produce plasmid pKS91.

SEQ ID NO:7 is a linker oligonucleotide used to insert variousrestriction enzyme sites into the plasmid pKS17 to form the plasmidpKS102.

SEQ ID NO:8 is the sequence of an oligonucleotide primer used in a PCRamplification of the soybean Cer3 gene for insertion into plasmid pKS67to form plasmid pKS100.

SEQ ID NO:9 is the sequence of an oligonucleotide primer used in a PCRamplification of the soybean Cer3 gene for insertion into plasmid pKS67to form plasmid pKS100.

SEQ ID NO:10 is the sequence of an oligonucleotide primer used in a PCRamplification of the soybean Cer3 gene for insertion into plasmid pKS67to form plasmid pKS100.

SEQ ID NO:11 is the sequence of an oligonucleotide primer used in a PCRamplification of the soybean Cer3 gene for insertion into plasmid pKS67to form plasmid pKS100.

SEQ ID NO:12 represents the 1× complementary repeat designatedELVISLIVES found in plasmids pKS106 and pKS124.

SEQ ID NO:13 represents the 2× complementary repeat designatedELVISLIVES found in plasmids pKS133.

SEQ ID NO:14 is the sequence of an oligonucleotide primer used in a PCRamplification of the ELVISLIVES complementary region.

SEQ ID NO:15 is the sequence of an oligonucleotide primer used in a PCRamplification of the ELVISLIVES complementary region.

SEQ ID NO:16 is the sequence of an oligonucleotide primer used in a PCRamplification of the soybean Fad2-1 gene to produce the 599 nucleotidefragment inserted into plasmid pKS106 to produce the plasmid pKS111.

SEQ ID NO:17 is the sequence of an oligonucleotide primer used in a PCRamplification of the soybean Fad2-1 gene to produce the 599 nucleotidefragment inserted into plasmid pKS106 to produce the plasmid pKS111.

SEQ ID NO:18 is the sequence of the common 5′ oligonucleotide primerused in a PCR amplification of the soybean Fad2-1 gene for use intesting size requirements for target sequences.

SEQ ID NO:19 is the sequence of a 3′ oligonucleotide primer used in aPCR amplification of the soybean Fad2-1 gene for production of the 25 bpfragment.

SEQ ID NO:20 is the sequence of a 3′ oligonucleotide primer used in aPCR amplification of the soybean Fad2-1 gene for production of the 75 bpfragment.

SEQ ID NO:21 is the sequence of a 3′ oligonucleotide primer used in aPCR amplification of the soybean Fad2-1 gene for production of the 150bp fragment.

SEQ ID NO:22 is the sequence of a 3′ oligonucleotide primer used in aPCR amplification of the soybean Fad2-1 gene for production of the 300bp fragment.

SEQ ID NO:23 is the sequence of a 3′ oligonucleotide primer used in aPCR amplification of the soybean Fad2-1 gene for production of the 600bp fragment.

SEQ ID NO:24 represents the 2× ELVISLIVES complementary repeat regionfrom pBS68 which contains 2× ELVISLIVES complementary regionssurrounding the 599 nucleotide Fad2-1 NotI fragment from pKS111 and a969 nucleotide fragment from a soybean delta-9 desaturase.

SEQ ID NO:25 is the sequence of a 5′ oligonucleotide primer used in aPCR amplification of the Lea promoter.

SEQ ID NO:26 is the sequence of a 3′ oligonucleotide primer used in aPCR amplification of the Lea promoter.

SEQ ID NO:27 is the sequence of a 5′ oligonucleotide primer used in aPCR amplification of the phaseolin 3′-end.

SEQ ID NO:28 is the sequence of a 3′ oligonucleotide primer used in aPCR amplification of the phaseolin 3′-end.

SEQ ID NO:29 represents the 2× ELVISLIVES complementary repeat regionfrom pKS149 that contains fragments from two soybean galactinol synthasegenes GAS1 and GAS2 (411 and 435 nucleotides, respectively). The regionis functionally attached to a late-soybean-embryo promoter (LEA) and aphaseolin 3′ terminator region. This entire region is then cloned intothe BamHI site of pKS136, which contains a 2× ELVISLIVES complementaryrepeat region controlled by a soybean Kti promoter and terminatorregion.

SEQ ID NO:30 represents the DNA sequence of the soybean galactinolsynthase gene GAS1.

SEQ ID NO:31 represents the putative translation product DNA sequence ofSEQ ID NO:30 the soybean galactinol synthase gene GAS1.

SEQ ID NO:32 represents the DNA sequence of the soybean galactinolsynthase gene GAS2.

SEQ ID NO:33 represents the putative translation product DNA sequence ofSEQ ID NO:32 the soybean galactinol synthase gene GAS2.

SEQ ID NO:34 represents the complementary region SHH3 from plasmidPHP17962, used in the construction of plasmid PHP17894 containing thephytoene desaturase fragment. The complementary regions are from 8-212and 305-509, respectively. Restriction endonuclease sites for EcoRV,KpnI, KspI, SphI, and NcoI can be used as cloning sites between thecomplementary regions.

SEQ ID NO:35 represents the DNA sequence of the soybean acetolactatesynthase (ALS) gene.

SEQ ID NO:36 is the sequence of a 3′ oligonucleotide primer used in aPCR amplification of the soybean Fad2-1 gene for production of the 50 bpfragment.

DETAILED DESCRIPTION OF THE INVENTION

In the context of this disclosure, a number of terms shall be utilized.

The term “host” refers to any organism, or cell thereof, whether humanor non-human into which a recombinant construct can be stably ortransiently introduced in order to reduce gene expression.

As used herein, an “isolated nucleic acid fragment” is a polymer of RNAor DNA that is single- or double-stranded, optionally containingsynthetic, non-natural or altered nucleotide bases. An isolated nucleicacid fragment in the form of a polymer of DNA may be comprised of one ormore segments of cDNA, genomic DNA or synthetic DNA. Nucleotides(usually found in their 5′-monophosphate form) are referred to by theirsingle letter designation as follows: “A” for adenylate ordeoxyadenylate (for RNA or DNA, respectively), “C” for cytidylate ordeoxycytidylate, “G” for guanylate or deoxyguanylate, “U” for uridylate,“T” for deoxythymidylate, “R” for purines (A or G), “Y” for pyrimidines(C or T), “K” for g or T, “H” for A or C or T, “I” for inosine, and “N”for any nucleotide.

The terms “subfragment that is functionally equivalent” and“functionally equivalent subfragment” are used interchangeably herein.These terms refer to a portion or subsequence of an isolated nucleicacid fragment in which the ability to alter gene expression or produce acertain phenotype is retained whether or not the fragment or subfragmentencodes an active enzyme. For example, the fragment or subfragment canbe used in the design of chimeric genes to produce the desired phenotypein a transformed plant. Chimeric genes can be designed for use inco-suppression or antisense by linking a nucleic acid fragment orsubfragment thereof, whether or not it encodes an active enzyme, in theappropriate orientation relative to a plant promoter sequence.

The terms “homology”, “homologous”, “substantially similar” and“corresponding substantially” are used interchangeably herein. Theyrefer to nucleic acid fragments wherein changes in one or morenucleotide bases does not affect the ability of the nucleic acidfragment to mediate gene expression or produce a certain phenotype.These terms also refer to modifications of the nucleic acid fragments ofthe instant invention such as deletion or insertion of one or morenucleotides that do not substantially alter the functional properties ofthe resulting nucleic acid fragment relative to the initial, unmodifiedfragment. It is therefore understood, as those skilled in the art willappreciate, that the invention encompasses more than the specificexemplary sequences.

Moreover, the skilled artisan recognizes that substantially similarnucleic acid sequences encompassed by this invention are also defined bytheir ability to hybridize, under moderately stringent conditions (forexample, 0.5×SSC, 0.1% SDS, 60° C.) with the sequences exemplifiedherein, or to any portion of the nucleotide sequences reported hereinand which are functionally equivalent to the promoter of the invention.Stringency conditions can be adjusted to screen for moderately similarfragments, such as homologous sequences from distantly relatedorganisms, to highly similar fragments, such as genes that duplicatefunctional enzymes from closely related organisms. Post-hybridizationwashes determine stringency conditions. One set of preferred conditionsinvolves a series of washes starting with 6×SSC, 0.5% SDS at roomtemperature for 15 min, then repeated with 2×SSC, 0.5% SDS at 45° C. for30 min, and then repeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30min. A more preferred set of stringent conditions involves the use ofhigher temperatures in which the washes are identical to those aboveexcept for the temperature of the final two 30 min washes in 0.2×SSC,0.5% SDS was increased to 60° C. Another preferred set of highlystringent conditions involves the use of two final washes in 0.1×SSC,0.1% SDS at 65° C.

With respect to the degree of substantial similarity between the target(endogenous) mRNA and the RNA region in the construct having homology tothe target mRNA, such sequences should be at least 25 nucleotides inlength, preferably at least 50 nucleotides in length, more preferably atleast 100 nucleotides in length, again more preferably at least 200nucleotides in length, and most preferably at least 300 nucleotides inlength; and should be at least 80% identical, preferably at least 85%identical, more preferably at least 90% identical, and most preferablyat least 95% identical.

Sequence alignments and percent similarity calculations may bedetermined using a variety of comparison methods designed to detecthomologous sequences including, but not limited to, the Megalign programof the LASARGENE bioinformatics computing suite (DNASTAR Inc., Madison,Wis.). Multiple alignment of the sequences are performed using theClustal method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153)with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10).Default parameters for pairwise alignments and calculation of percentidentity of protein sequences using the Clustal method are KTUPLE=1, GAPPENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids theseparameters are KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. A “foreign” gene refers to a gene not normally found in thehost organism, but that is introduced into the host organism by genetransfer. Foreign genes can comprise native genes inserted into anon-native organism, or chimeric genes. A “transgene” is a gene that hasbeen introduced into the genome by a transformation procedure.

The term “essential plant genes” as used herein refers to genes encodinga product that is required for normal plant growth, development, and/orviability. In addition to ALS, examples of essential plant genes wouldinclude, but not be limited to, rate-limiting enzymes in amino acid,nucleic acid, or lipid biosynthesis. It is also believed that many geneswith unknown function may be essential. Suppression of essential plantgenes by chemical or genetic means will result in altered growth and/ordevelopment. If an essential gene is unique in the genome of the plant,suppression may lead to plant death, which is the basis of many plantherbicides.

“Coding sequence” refers to a DNA sequence that codes for a specificamino acid sequence. “Regulatory sequences” refer to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may include, butare not limited to, promoters, translation leader sequences, introns,and polyadenylation recognition sequences.

“Promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. The promoter sequenceconsists of proximal and more distal upstream elements, the latterelements often referred to as enhancers. Accordingly, an “enhancer” is aDNA sequence which can stimulate promoter activity and may be an innateelement of the promoter or a heterologous element inserted to enhancethe level or tissue-specificity of a promoter. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. It is understood by those skilled inthe art that different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental conditions. Promoters whichcause a gene to be expressed in most cell types at most times arecommonly referred to as “constitutive promoters”. New promoters ofvarious types useful in plant cells are constantly being discovered;numerous examples may be found in the compilation by Okamuro andGoldberg, (1989) Biochemistry of Plants 15:1-82. It is furtherrecognized that since in most cases the exact boundaries of regulatorysequences have not been completely defined, DNA fragments of somevariation may have identical promoter activity.

An “intron” is an intervening sequence in a gene that does not encode aportion of the protein sequence. Thus, such sequences are transcribedinto RNA but are then excised and are not translated. The term is alsoused for the excised RNA sequences. An “exon” is a portion of thesequence of a gene that is transcribed and is found in the maturemessenger RNA derived from the gene, but is not necessarily a part ofthe sequence that encodes the final gene product.

The “translation leader sequence” refers to a DNA sequence locatedbetween the promoter sequence of a gene and the coding sequence. Thetranslation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency. Examples of translation leadersequences have been described (Turner, R. and Foster, G. D. (1995)Molecular Biotechnology 3:225). The “3′ non-coding sequences” refer toDNA sequences located downstream of a coding sequence and includepolyadenylation recognition sequences and other sequences encodingregulatory signals capable of affecting mRNA processing or geneexpression. The polyadenylation signal is usually characterized byaffecting the addition of polyadenylic acid tracts to the 3′ end of themRNA precursor. The use of different 3′ non-coding sequences isexemplified by Ingelbrecht et al, (1989) Plant Cell 1:671-680.

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from post-transcriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated into proteinby the cell. “cDNA” refers to a DNA that is complementary to andsynthesized from a mRNA template using the enzyme reverse transcriptase.The cDNA can be single-stranded or converted into the double-strandedform using the Klenow fragment of DNA polymerase I. “Sense” RNA refersto RNA transcript that includes the mRNA and can be translated intoprotein within a cell or in vitro. “Antisense RNA” refers to an RNAtranscript that is complementary to all or part of a target primarytranscript or mRNA and that blocks the expression of a target gene (U.S.Pat. No. 5,107,065). The complementarity of an antisense RNA may be withany part of the specific gene transcript, i.e., at the 5′ non-codingsequence, 3′ non-coding sequence, introns, or the coding sequence.“Functional RNA” refers to antisense RNA, ribozyme RNA, or other RNAthat may not be translated but yet has an effect on cellular processes.The terms “complement” and “reverse complement” are used interchangeablyherein with respect to mRNA transcripts, and are meant to define theantisense RNA of the message.

The term “target mRNA” refers to any mRNA whose expression in the hostis to be reduced.

The term “endogenous RNA” refers to any RNA which is encoded by anynucleic acid sequence present in the genome of the host prior totransformation with the recombinant construct of the present invention,whether naturally-occurring or non-naturally occurring, i.e., introducedby recombinant means, mutagenesis, etc.

The term “non-naturally occurring” means artificial, not consistent withwhat is normally found in nature.

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis regulated by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of regulating the expressionof that coding sequence (i.e., that the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in a sense or antisenseorientation. In another example, the complementary RNA regions of theinvention can be operably linked, either directly or indirectly, 5′ tothe target mRNA, or 3′ to the target mRNA, or within the target mRNA, ora first complementary region is 5′ and its complement is 3′ to thetarget mRNA.

The term “expression”, as used herein, refers to the production of afunctional end-product. Expression of a gene involves transcription ofthe gene and translation of the mRNA into a precursor or mature protein.“Antisense inhibition” refers to the production of antisense RNAtranscripts capable of suppressing the expression of the target protein.“Co-suppression” refers to the production of sense RNA transcriptscapable of suppressing the expression of identical or substantiallysimilar foreign or endogenous genes (U.S. Pat. No. 5,231,020).

“Mature” protein refers to a post-translationally processed polypeptide;i.e., one from which any pre- or propeptides present in the primarytranslation product have been removed. “Precursor” protein refers to theprimary product of translation of mRNA; i.e., with pre- and propeptidesstill present. Pre- and propeptides may be but are not limited tointracellular localization signals.

“Stable transformation” refers to the transfer of a nucleic acidfragment into a genome of a host organism, including both nuclear andorganellar genomes, resulting in genetically stable inheritance. Incontrast, “transient transformation” refers to the transfer of a nucleicacid fragment into the nucleus, or DNA-containing organelle, of a hostorganism resulting in gene expression without integration or stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” organisms. The preferredmethod of cell transformation of rice, corn and other monocots is theuse of particle-accelerated or “gene gun” transformation technology(Klein et al, (1987) Nature (London) 327:70-73; U.S. Pat. No.4,945,050), or an Agrobacterium-mediated method using an appropriate Tiplasmid containing the transgene (Ishida Y. et al, 1996, Nature Biotech.14:745-750).

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook, J.,Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual;Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989(hereinafter “Sambrook”).

The term “recombinant” refers to an artificial combination of twootherwise separated segments of sequence, e.g., by chemical synthesis orby the manipulation of isolated segments of nucleic acids by geneticengineering techniques.

“PCR” or “Polymerase Chain Reaction” is a technique for the synthesis oflarge quantities of specific DNA segments, consists of a series ofrepetitive cycles (Perkin Elmer Cetus Instruments, Norwalk, Conn.).Typically, the double stranded DNA is heat denatured, the two primerscomplementary to the 3′ boundaries of the target segment are annealed atlow temperature and then extended at an intermediate temperature. Oneset of these three consecutive steps is referred to as a cycle.

The terms “recombinant construct”, “expression construct” and“recombinant expression construct” are used interchangeably herein. Suchconstruct may be itself or may be used in conjunction with a vector. Ifa vector is used then the choice of vector is dependent upon the methodthat will be used to transform host plants as is well known to thoseskilled in the art. For example, a plasmid vector can be used. Theskilled artisan is well aware of the genetic elements that must bepresent on the vector in order to successfully transform, select andpropagate host cells comprising any of the isolated nucleic acidfragments of the invention. The skilled artisan will also recognize thatdifferent independent transformation events will result in differentlevels and patterns of expression (Jones et al, (1985) EMBO J.4:2411-2418; De Almeida et al, (1989) Mol. Gen. Genetics 218:78-86), andthus that multiple events must be screened in order to obtain linesdisplaying the desired expression level and pattern. Such screening maybe accomplished by Southern analysis of DNA, Northern analysis of mRNAexpression, Western analysis of protein expression, or phenotypicanalysis.

Co-suppression constructs in plants previously have been designed byfocusing on overexpression of a nucleic acid sequence having homology toan endogenous mRNA, in the sense orientation, which results in thereduction of all RNA having homology to the overexpressed sequence (seeVaucheret et al (1998) Plant J 16:651-659; and Gura (2000) Nature404:804-808). The overall efficiency of this phenomenon is low, and theextent of the RNA reduction is widely variable. Recent work hasdescribed the use of “hairpin” structures that incorporate all, or part,of an mRNA encoding sequence in a complementary orientation that resultsin a potential “stem-loop” structure for the expressed RNA (PCTPublication WO 99/53050 published on Oct. 21, 1999). This increases thefrequency of co-suppression in the recovered transgenic plants. Anothervariation describes the use of plant viral sequences to direct thesuppression, or “silencing”, of proximal mRNA encoding sequences (PCTPublication WO 98/36083 published on Aug. 20, 1998). Both of theseco-suppressing phenomena have not been elucidated mechanistically,although recent genetic evidence has begun to unravel this complexsituation (Elmayan et al (1998) Plant Cell 10:1747-1757).

Surprisingly and unexpectedly, it has been found that suitable nucleicacid sequences and their reverse complement can be used to alter theexpression of any homologous, endogenous RNA (i.e., the target RNA)which is in proximity to the suitable nucleic acid sequence and itsreverse complement. As is discussed below, the suitable nucleic acidsequence and its reverse complement can be either unrelated to anyendogenous RNA in the host or can be encoded by any nucleic acidsequence in the genome of the host provided that nucleic acid sequencedoes not encode any target mRNA or any sequence that is substantiallysimilar to the target mRNA.

Thus, the present invention presents a very efficient and robustapproach to achieving single, or multiple, gene co-suppression usingsingle plasmid transformation. As is discussed in greater detail below,the constructs are composed of promoters linked to mRNA(s) codingregions, or fragments thereof, that are targeted for suppression, andshort complementary sequences that are unrelated to the targets. Thecomplementary sequences can be oriented both 5′, both 3′, or on eitherside of the target sequence. The complementary sequences are preferredto be about 40-50 nucleotides in length, or more preferably 50-100nucleotides in length, or most preferably at least or greater than100-300 nucleotides. The complementary sequences are unrelated to thetarget, but can come from any other source. Preferred embodiments ofthese sequences include, but are not limited to, plant sequences,bacterial sequences, animal sequences, viral or phage sequences, orcompletely artificial, i.e. non-naturally occurring, sequences not knownto occur in any organism (see “ELVISLIVES” below). All sequences can becompared to other known sequences, or each other, using any one of anumber of sequence alignment programs as set forth below in Example 4.

The term “high degree of frequency” as used herein, with respect to thesuppression efficiency, refers to the percentage of transformed linesthat exhibit the target suppressed phenotype. High frequency percentagesare expected to be in a range of at least 15-95% and any integerpercentage found within the range. Preferred embodiments would includeat least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, and 95%.

The present invention concerns a recombinant construct comprising apromoter operably linked to a DNA sequence which, when expressed by ahost produces an RNA having:

(a) homology to at least one target mRNA expressed by the host,

(b) two complementary RNA regions which are unrelated to any endogenousRNA in the host, and which are in proximity to (a),

wherein the expressed RNA reduces the expression of the target mRNA orany substantially similar endogenous mRNA.

In another aspect the present invention concerns a recombinant constructcomprising a promoter operably linked to a DNA sequence which, whenexpressed by a host produces an RNA having:

(a) homology to at least one target mRNA expressed by the host,

(b) two complementary RNA regions which are encoded by any nucleic acidsequence in the genome of the host provided that said sequence does notencode the target mRNA or any sequence that is substantially similar tothe target mRNA and said regions are in proximity to (a),

wherein the expressed RNA reduces the expression of the target mRNA orany substantially similar endogenous mRNA.

Any promoter can be used to practice the invention. There can bementioned a beta-conglycinin promoter, a Kunitz soybean TrypsinInhibitor (KSTI or Kti) promoter, a Gly m Bd 28K promoter, T7 promoter,a 35S promoter and a beta-phaseolin promoter. The preferred promoter isthat of the α′-subunit of beta-conglycinin (referred to herein as thebeta-conglycinin promoter). Co-suppressed plants that containrecombinant expression constructs with the promoter of the α′-subunit ofbeta-conglycinin will often exhibit suppression of both the α and α′subunits of beta-congylcinin (as described in PCT Publication No. WO97/47731, published on Dec. 18, 1997, the disclosure of which is herebyincorporated by reference). Particularly preferred promoters are thosethat allow seed-specific expression. This may be especially useful sinceseeds are the primary source consumable protein and oil, and also sinceseed-specific expression will avoid any potential deleterious effect innon-seed tissues.

Examples of seed-specific promoters include, but are not limited to, thepromoters of seed storage proteins, which can represent up to 90% oftotal seed protein in many plants. The seed storage proteins arestrictly regulated, being expressed almost exclusively in seeds in ahighly tissue-specific and stage-specific manner (Higgins et al, (1984)Ann. Rev. Plant Physiol. 35:191-221; Goldberg et al, (1989) Cell56:149-160). Moreover, different seed storage proteins may be expressedat different stages of seed development.

Expression of seed-specific genes has been studied in great detail (Seereviews by Goldberg et al, (1989) Cell 56:149-160 and Higgins et al,(1984) Ann. Rev. Plant Physiol. 35:191-221). There are currentlynumerous examples of seed-specific expression of seed storage proteingenes in transgenic dicotyledonous plants. These include genes fromdicotyledonous plants for bean β-phaseolin (Sengupta-Gopalan et al,(1985) Proc. Natl. Acad. Sci. USA 82: 3320-3324; Hoffman et al, (1988)Plant Mol. Biol. 11: 717-729), bean lectin (Voelker et al, (1987) EMBOJ. 6: 3571-3577), soybean lectin (Okamuro et al, (1986) Proc. Natl.Acad. Sci. USA 83: 8240-8244), soybean Kunitz trypsin inhibitor(Perez-Grau et al, (1989) Plant Cell 1: 095-1109), soybean b-conglycinin(Beachy et al, (1985) EMBO J. 4: 3047-3053; pea vicilin (Higgins et al,(1988) Plant Mol. Biol. 11:683-695), pea convicilin (Newbigin et al,(1990) Planta 180:461-470), pea legumin (Shirsat et al, (1989) Mol. Gen.Genetics 215:326-331); rapeseed napin (Radke et al, (1988) Theor. Appl.Genet. 75:685-694) as well as genes from monocotyledonous plants such asfor maize 15 kD zein (Hoffman et al, (1987) EMBO J. 6:3213-3221), maize18 kD oleosin (Lee at al., (1991) Proc. Natl. Acad. Sci. USA88:6181-6185), barley β-hordein (Marris et al, (1988) Plant Mol. Biol.10:359-366) and wheat glutenin (Colot et al, (1987) EMBO J.6:3559-3564). Moreover, promoters of seed-specific genes operably linkedto heterologous coding sequences in chimeric gene constructs alsomaintain their temporal and spatial expression pattern in transgenicplants. Such examples include use of Arabidopsis thaliana 2S seedstorage protein gene promoter to express enkephalin peptides inArabidopsis and Brassica napus seeds (Vandekerckhove et al, (1989)Bio/Technology 7:929-932), bean lectin and bean β-phaseolin promoters toexpress luciferase (Riggs et al, (1989) Plant Sci. 63:47-57), and wheatglutenin promoters to express chloramphenicol acetyl transferase (Colotet al, (1987) EMBO J. 6:3559-3564).

As was noted above any type of promoter such as constitutive,tissue-preferred or inducible promoters can be used to practice theinvention. Examples of constitutive promoters include the cauliflowermosaivirus (CaMV) 35S transcription initiation region, the 1′- or2′-promoter derived from T-DNA of Agrobacterium tumefaciens, theubiquitin 1 promoter, the Smas promoter, the cinnamyl alcoholdehydrogenase promoter (U.S. Pat. No. 5,683,439), the Nos promoter, thepEmu promoter, the rubisco promoter, the GRP1-8 promoter and othertranscription initiation regions from various plant genes known to thoseof skill.

Examples of inducible promoters are the Adh1 promoter which is inducibleby hypoxia or cold stress, the Hsp70 promoter which is inducible by heatstress, and the PPDK promoter which is inducible by light. Also usefulare promoters that are chemically inducible.

Examples of promoters under developmental control include promoters thatinitiate transcription preferentially in certain tissues, such asleaves, roots, fruit, seeds, or flowers. An exemplary promoter is theanther specific promoter 5126 (U.S. Pat. Nos. 5,689,049 and 5,689,051).In addition to those mentioned above, other examples of seed-specificpromoters include, but are not limited to, 27 kD gamma zein promoter andwaxy promoter, Boronat, A., Martinez, M. C., Reina, M., Puigdomenech, P.and Palau, J.; Isolation and sequencing of a 28 kD glutelin-2 gene frommaize: Common elements in the 5′ flanking regions among zein andglutelin genes; Plant Sci. 47, 95-102 (1986) and Reina, M., Ponte, I.,Guillen, P., Boronat, A. and Palau, J., Sequence analysis of a genomicclone encoding a Zc2 protein from Zea mays W64 A, Nucleic Acids Res. 18(21), 6426 (1990). See the following site relating to the waxy promoter:Kloesgen, R. B., Gierl, A., Schwarz-Sommer, Z. S. and Saedler, H.,Molecular analysis of the waxy locus of Zea mays, Mol. Gen. Genet. 203,237-244 (1986). Promoters that express in the embryo, pericarp, andendosperm are disclosed in PCT Application No. WO 00/11177 publishedMar. 2, 2000, and PCT Application No. WO 00/12733 published Mar. 9,2000. The disclosures each of these are incorporated herein by referencein their entirety.

Either heterologous or non-heterologous (i.e., endogenous) promoters canbe used to practice the invention.

The promoter is then operably linked using conventional means well knownto those skilled in the art to a DNA sequence which, when expressed by ahost produces an RNA meeting certain criteria.

The host can be any organism, or cell thereof, into which therecombinant construct of this invention can be stably or transientlyintroduced in order to alter gene expression. Examples of suitable hostsinclude, but are not limited to, a plant, animal, protozoan, bacterium,virus or fungus. The plant may be a monocot, dicot or gymnosperm; theanimal may be a vertebrate or invertebrate. Preferred microbes are thoseused in agriculture or by industry. Fungi include organisms in both themold and yeast morphologies.

Plants include Arabidopsis; field crops (e.g., alfalfa, barley, bean,corn, cotton, flax, pea, rape, rice, rye, safflower, sorghum, soybean,sunflower, tobacco, and wheat); vegetable crops (e.g., asparagus, beet,broccoli, cabbage, carrot, cauliflower, celery, cucumber, eggplant,lettuce, onion, pepper, potato, pumpkin, radish, spinach, squash, taro,tomato, and zucchini); fruit and nut crops (e.g., almond, apple,apricot, banana, blackberry, blueberry, cacao, cherry, coconut,cranberry, date, fajoa, filbert, grape, grapefruit, guava, kiwi, lemon,lime, mango, melon, nectarine, orange, papaya, passion fruit, peach,peanut, pear, pineapple, pistachio, plum, raspberry, strawberry,tangerine, walnut, and watermelon); etc.

Examples of human or non-human vertebrate animals include mammals, fish,cattle, goat, pig, sheep, rodent, hamster, mouse, rat, guinea pigs,rabbits, and primate; invertebrate animals include nematodes, otherworms, Drosophila, and other insects. Representative orders of insectsinclude Coleoptera, Diptera, Lepidoptera, and Homoptera.

The DNA sequence expressed by the host produces an RNA having:

(a) homology to at least one target mRNA expressed by the host;

(b1) two complementary RNA regions which are unrelated to any endogenousRNA in the host, and which are in proximity to the target mRNA, whereinthe expressed RNA reduces the expression of the target RNA or anysubstantially similar endogenous mRNA, or

(b2) two complementary RNA regions which are encoded by any nucleic acidsequence in the genome of the host provided that said sequence does notencode the target mRNA or any sequence that is substantially similar tothe target mRNA and said regions are in proximity to (a).

The complementary RNA regions may comprise any of the following:

(a) any nucleic acid sequence not normally present in the genome of ahost, i.e, are not related to any endogenous RNA in the host; or

(b) any nucleic acid sequence in the genome of the host which encodesthe complementary regions provided that said sequence does not encodethe target mRNA or any sequence that is substantially similar to thetarget mRNA.

With respect to (a) any nucleic acid sequence not normally present inthe genome of a host, the RNA region or regions which are unrelated toany endogenous RNA in the host may comprise a synthetic, non-naturallyoccurring RNA sequence. In still a further aspect, these RNA region orregions, optionally, may or may not comprise plant viral RNA.

With respect to (b) any nucleic acid sequence in the genome of the hostwhich encodes the complementary regions provided that said sequence doesnot encode the target mRNA or any sequence that is substantially similarto the target mRNA, this sequence comprises transcribed ornon-transcribed nucleic acid sequences which may be present in thegenome of the host, i.e., this sequence may or may not be expressed bythe host.

The complementary RNA regions described herein are in proximity to thetarget mRNA. The term “in proximity” means that the complementaryregions are operably linked 5′ to the target mRNA, or 3′ to the targetmRNA, or within the target mRNA, or 5′ and 3′ to the target mRNA, i.e.,the complementary regions or sequences can be found on either end of thetarget mRNA.

The complementary RNA regions can be any size that is suitable foraltering the expression of the target mRNA. The complementary sequencesare preferred to be about 40-50 nucleotides in length, or morepreferably 50-100 nucleotides in length, or most preferably greater than100-300 nucleotides. These complementary sequences can be synthesizedusing conventional means well known to those skilled in the art.

Examples of suitable complementary RNA regions which can be used topractice the invention include, but are not limited to, SEQ ID NO:12 and13, bacterial sequences, jellyfish sequences, or any artificial ornaturally occurring sequences.

In another embodiment this invention concerns a method for reducingexpression of a target mRNA or any substantially similar endogenous mRNAwhich comprises:

(a) introducing into a host any of the recombinant constructs discussedherein, and

(b) selecting hosts which have reduced expression of the target mRNA orany substantially similar endogenous mRNA.

Transformation methods are discussed above and are well known to thoseskilled in the art.

Selection of the host having the desired phenotype will depend upon thetarget mRNA whose expression is being altered. As was noted above, thetarget mRNA may be any mRNA whose expression in the host is to bealtered. Typically, it should share homology with the RNA produced bythe host transformed with a recombinant construct of the invention. Theexpression of more than one target mRNA may be reduced provided thatthese targets share homology with the RNA produced by the hosttransformed with a recombinant construct of the invention.

In still another embodiment, this invention concerns a recombinantconstruct comprising an RNA in lieu of a DNA sequence. Thus, this RNAcomprises:

(a) homology to at least one target mRNA expressed by a host,

(b) two complementary RNA regions which are unrelated to any endogenousRNA in the host, and which are in proximity to (a),

wherein the RNA, when introduced into the host, reduces the expressionof the target mRNA or any substantially similar endogenous mRNA.

In another aspect, this invention concerns a recombinant constructcomprising an RNA in lieu of a DNA sequence in which the RNA comprises:

(a) homology to at least one target mRNA expressed by a host,

(b) two complementary RNA regions which are encoded by any nucleic acidsequence in the genome of the host provided that said sequence does notencode the target mRNA or any sequence that is substantially similar tothe target mRNA and which regions are in proximity to (a),

wherein the RNA, when introduced into the host, reduces the expressionof the target mRNA or any substantially similar endogenous mRNA.

As was discussed above the complementary RNA regions may comprise any ofthe following:

(a) any nucleic acid sequence not normally present in the genome of ahost, i.e, are not related to any endogenous RNA in the host; or

(b) any nucleic acid sequence in the genome of the host which encodesthe complementary regions provided that said sequence does not encodethe target mRNA or any sequence that is substantially similar to thetarget mRNA.

With respect to (a) any nucleic acid sequence not normally present inthe genome of a host, the RNA region or regions which are unrelated toany endogenous RNA in the host may comprise a synthetic, non-naturallyoccurring RNA sequence. In still a further aspect, these RNA region orregions, optionally, may or may not comprise plant viral RNA.

With respect to (b) any nucleic acid sequence in the genome of the hostwhich encodes the complementary regions provided that said sequence doesnot encode the target mRNA or any sequence that is substantially similarto the target mRNA, this sequence comprises transcribed ornon-transcribed nucleic acid sequences which may be present in thegenome of the host.

The complementary RNA regions described herein are in proximity to thetarget mRNA. The term “in proximity” means that the complementaryregions are operably linked 5′ to the target mRNA, or 3′ to the targetmRNA, or within the target mRNAS, or 5′ and 3′ to the target mRNA, i.e.,the complementary regions or sequences can be found on either end of thetarget mRNA.

In addition, these RNAs can be used in a method for reducing expressionof a target mRNA or any substantially similar endogenous mRNA whichcomprises:

(a) introducing into a host any of the RNAs described herein; and

(b) selecting hosts which have reduced expression of the target mRNA orany substantially similar endogenous mRNA.

In still a further aspect, the present invention concerns a method foridentifying or screening an essential plant gene which comprises:

(a) transforming a plant cell with a recombinant construct comprising aconstitutive promoter wherein said construct is capable of reducingexpression of an essential plant gene with a high degree of frequency;

(b) quantifying all transformed plant cells from step (a);

(c) quantifying all transformed plant cells from a control which doesnot reduce expression of an essential plant gene; and

(d) comparing the quantification of transformed plant cells selectedfrom step (b) with the quantification of transformed plants cellsselected from step (c) wherein the quantification of transformed plantscells selected from step (c) should substantially exceed thequantification of transformed plant cells selected from step (b).

Any essential plant gene can be identified or screened using the methodof the invention. An important aspect of this method is the use of aconstitutive promoter and a recombinant construct capable of reducingexpression of an essential plant gene with a high degree of frequency.

Essential plant genes are defined above.

Constitutive promoters are defined above. Preferably, the constitutivepromoter is a high level or strong constitutive promoter whereinexpression of the gene under the control of the promoter results inproduction of high levels of mRNA.

Any recombinant construct comprising a constitutive promoter which iscapable of reducing expression of an essential plant gene with a highdegree of frequency can be used to practice the invention. In apreferred embodiment, the recombinant construct can be any of therecombinant constructs of the invention comprising a promoter operablylinked to a DNA sequence provided that the promoter is a constitutivepromoter. The term high degree of frequency is defined above.

Any plant cells can be transformed using standard transformation methodsas described above.

The number of plant cells transformed with a recombinant constructcomprising a constitutive promoter wherein the recombinant construct isdesigned to reduce expression of an essential plant gene is quantifiedand compared to the number of plant cells transformed using a control inwhich expression of the essential plant gene is not reduced. If thenumber of plant cells transformed with the control substantially exceedsthe number of plant cells transformed with the recombinant constructdesigned to reduce expression of an essential plant gene, then anessential plant gene has been identified/screened. By “substantiallyexceeds”, it is meant at least a five-fold difference and, preferably, aten-fold difference. Also preferred would be a 4-fold, 6-fold, 7-fold,8-fold, 9-fold, or greater than a 10-fold difference. Thus, the numberof plant cells transformed with the control should be at least five-foldgreater than the number of plant cells transformed with the recombinantconstruct designed to reduce expression of an essential plant gene.

EXAMPLES

The present invention is further defined in the following Examples, inwhich all parts and percentages are by weight and degrees are Celsius,unless otherwise stated. It should be understood that these Examples,while indicating preferred embodiments of the invention, are given byway of illustration only. From the above discussion and these Examples,one skilled in the art can ascertain the essential characteristics ofthis invention, and without departing from the spirit and scope thereof,can make various changes and modifications of the invention to adapt itto various usages and conditions. The disclosures contained within thereferences used herein are hereby incorporated by reference.

Example 1 Transformation of Somatic Soybean Embryo Cultures

Generic Stable Soybean Transformation Protocol:

Soybean embryogenic suspension cultures are maintained in 35 ml liquidmedia (SB55 or SBP6) on a rotary shaker, 150 rpm, at 28° C. with mixedfluorescent and incandescent lights on a 16:8 h day/night schedule.Cultures are subcultured every four weeks by inoculating approximately35 mg of tissue into 35 ml of liquid medium. TABLE 1 Stock Solutions(g/L): MS Sulfate 100X Stock MgSO₄7H₂O 37.0 MnSO₄H₂O 1.69 ZnSO₄7H₂O 0.86CuSO₄5H₂O 0.0025 MS Halides 100X Stock CaCl₂ 2H₂O 44.0 KI 0.083CoCl₂6H₂0 0.00125 KH₂PO₄ 17.0 H₃BO₃ 0.62 Na₂MoO₄2H₂O 0.025 MS FeEDTA100X Stock Na₂EDTA 3.724 FeSO₄7H₂O 2.784 B5 Vitamin Stock 10 gm-inositol 100 mg nicotinic acid 100 mg pyridoxine HCl 1 g thiamine SB55(per Liter, pH 5.7) 10 ml each MS stocks 1 ml B5 Vitamin stock 0.8 gNH₄NO₃ 3.033 g KNO₃ 1 ml 2,4-D (10 mg/mL stock) 60 g sucrose 0.667 gasparagine SBP6 same as SB55 except 0.5 ml 2,4-D SB103 (per Liter, pH5.7) 1X MS Salts 6% maltose 750 mg MgCl₂ 0.2% Gelrite SB71-1 (per Liter,pH 5.7) 1X B5 salts 1 ml B5 vitamin stock 3% sucrose 750 mg MgCl₂ 0.2%Gelrite

Soybean embryogenic suspension cultures are transformed with pTC3 by themethod of particle gun bombardment (Klein et al (1987) Nature 327:70). ADuPont Biolistic PDS1000/HE instrument (helium retrofit) is used forthese transformations.

To 50 ml of a 60 mg/ml 1 μm gold particle suspension is added (inorder); 5 μl DNA (1 μg/μl), 20 μl spermidine (0.1 M), and 50 μl CaCl₂(2.5 M). The particle preparation is agitated for 3 min, spun in amicrofuge for 10 sec and the supernatant removed. The DNA-coatedparticles are then washed once in 400 μl 70% ethanol and re suspended in40 μl of anhydrous ethanol. The DNA/particle suspension is sonicatedthree times for 1 sec each. Five μl of the DNA-coated gold particles arethen loaded on each macro carrier disk. For selection, a plasmidconferring resistance to hygromycin phosphotransferase (HPT) may beco-bombarded with the silencing construct of interest.

Approximately 300-400 mg of a four week old suspension culture is placedin an empty 60×15 mm petri dish and the residual liquid removed from thetissue with a pipette. For each transformation experiment, approximately5-10 plates of tissue are normally bombarded. Membrane rupture pressureis set at 1000 psi and the chamber is evacuated to a vacuum of 28 inchesof mercury. The tissue is placed approximately 3.5 inches away from theretaining screen and bombarded three times. Following bombardment, thetissue is placed back into liquid and cultured as described above.

Eleven days post bombardment, the liquid media is exchanged with freshSB55 containing 50 mg/ml hygromycin. The selective media is refreshedweekly. Seven weeks post bombardment, green, transformed tissue isobserved growing from untransformed, necrotic embryogenic clusters.Isolated green tissue is removed and inoculated into individual flasksto generate new, clonally propagated, transformed embryogenic suspensioncultures. Thus each new line is treated as an independent transformationevent. These suspensions can then be maintained as suspensions ofembryos maintained in an immature developmental stage or regeneratedinto whole plants by maturation and germination of individual somaticembryos.

Independent lines of transformed embryogenic clusters are removed fromliquid culture and placed on a solid agar media (SB103) containing nohormones or antibiotics. Embryos are cultured for four weeks at 26° C.with mixed fluorescent and incandescent lights on a 16:8 h day/nightschedule. During this period, individual embryos are removed from theclusters and screened for alterations in their fatty acid compositions(Example 3). Co-suppression of Fad2 results in a reduction ofpolyunsaturated fatty acids and an increase in oleic acid content.

It should be noted that any detectable phenotype, resulting from theco-suppression of a target gene, can be screened at this stage. Thiswould include, but not be limited to, alterations in protein content,carbohydrate content, growth rate, viability, or the ability to developnormally into a soybean plant.

Example 2 Transformation of Maize

Generic Stable Maize Transformation Protocol:

Transformation of plasmid DNA in Hi-II strains of maize follows thestandard Hi-II bombardment transformation protocol (Songstad D. D. etal, (1996) In Vitro Cell Dev. Biol. Plant 32:179-183). Cells aretransformed by culturing maize immature embryos (approximately 1-1.5 mmin length) onto 560P medium containing N6 salts, Erikkson's vitamins,0.69 g/l proline, 2 mg/l 2,4-D and 3% sucrose. After 4-5 days ofincubation in the dark at 28° C., embryos are removed from 560P mediumand cultured, scutellum up, onto 560Y medium which is equivalent to 560Pbut contains 12% sucrose. Embryos are allowed to acclimate to thismedium for 3 h prior to transformation. The scutellar surface of theimmature embryos is targeted using particle bombardment with either amixture containing UBI:moPAT:pinII+UBI:GUS:pinII plasmids, or with acombination of these two plasmids plus any one of the constructs of thepresent invention (UBI is the ubiquitin-1 promoter, Christensen et al(1989) Plant Mol Bio 12:619-632; moPAT refers to a “monocot-optimizedphosphinothricin acyltransferase” gene conferring resistance to theherbicide glufosinate ammonium, referenced in PCT Application No. WO98/30701 published on Jul. 16, 1998; the pinII (proteinase inhibitor)terminator is described in An et al (1989) Plant Cell 1:115-122; and theGUS gene (beta-glucuronidase) is described in Jefferson et al (1986)PNAS 83:8447-8451). Embryos are transformed using the PDS-1000 HeliumGun from Bio-Rad at one shot per sample using 650PSI rupture disks. DNAdelivered per shot averages about 0.1667 ug. An equal number of embryosper ear are bombarded with either the control DNA (PAT/GUS) or themixture of control with any one of the constructs of the presentinvention. Following bombardment, all embryos are cultured andmaintained on 560L medium (N6 salts, Eriksson's vitamins, 0.5 mg/lthiamine, 20 g/l sucrose, 1 mg/l 2,4-D, 2.88 g/l proline, 2.0 g/lgelrite, and 8.5 mg/l silver nitrate). After 2-7 days post-bombardment,all the embryos from both treatments are transferred onto N6-basedmedium containing 3 mg/l bialaphos Pioneer 560P medium described above,with no proline and with 3 mg/l bialaphos). Plates are maintained at 28°C. in the dark and are observed for colony recovery with transfers tofresh medium occurring every two weeks.

Transient Maize Assays:

High type II callus is maintained by subculturing onto fresh 560P mediumevery two weeks. Healthy callus is pushed through a 0.77 mm² nylon meshand resuspended in MS culture medium with 2 mg/l 2,4-D at a density of 3grams of tissue/40 ml medium. The cell suspension are then pipetted in 4ml aliquots (each containing approximately 300 mg of cells) onto glassfilter papers for bombardment using a vacuum apparatus. These filtersare then placed on 560P medium and cultured in the dark at 26° C. After2-4 days the filters are removed from the culture medium and excessliquid is removed using a vacuum apparatus. Filters with cells are thenshot (using the DuPont Biolistics PDS1000/He gun) according toestablished methods (see example above) using 1 μm gold particles and650 psi rupture disks. Immediately after bombardment filters arereturned to 560P culture medium and cultured in the dark at 26° C. AllDNA's are adjusted to obtain a final concentration of 1 μg/totalDNA/particle prep tube (6 shots). The typical experiment is shot asfollows:

-   -   GUS DNA+control DNA+Luciferase DNA    -   GUS DNA+silencing construct DNA+Luciferase DNA

Two days after bombardment cells are scraped from filters and protein isextracted, and enzyme activity is determined, using the luciferaseassays outlined in the Dual-Luciferase Reporter Assay protocol (PromegaCorp., Madison, Wis.). The same extract is also used to performfluorometric GUS assays using the protocol of Rao and Flynn (1990)Biotechniques 8:38-40. Data presented in Example 8 below is plotted asthe ratio of GUS/Luciferase units.

Example 3 The Phenotype of Transgenic Soybean Somatic Embryos isPredictive of Seed Phenotypes from Resultant Regenerated Plants

Mature somatic soybean embryos are a good model for zygotic embryos.While in the globular embryo state in liquid culture, somatic soybeanembryos contain very low amounts of triacylglycerol or storage proteinstypical of maturing, zygotic soybean embryos. At this developmentalstage, the ratio of total triacylglyceride to total polar lipid(phospholipids and glycolipid) is about 1:4, as is typical of zygoticsoybean embryos at the developmental stage from which the somatic embryoculture was initiated. At the globular stage as well, the mRNAs for theprominent seed proteins, α′ subunit of β-conglycinin, kunitz trypsininhibitor 3, and seed lectin are essentially absent. Upon transfer tohormone-free media to allow differentiation to the maturing somaticembryo state, triacylglycerol becomes the most abundant lipid class. Aswell, mRNAs for α′-subunit of β-conglycinin, kunitz trypsin inhibitor 3and seed lectin become very abundant messages in the total mRNApopulation. On this basis somatic soybean embryo system behaves verysimilarly to maturing zygotic soybean embryos in vivo, and is thereforea good and rapid model system for analyzing the phenotypic effects ofmodifying the expression of genes in the fatty acid biosynthesispathway.

Most importantly, the model system is also predictive of the fatty acidcomposition of seeds from plants derived from transgenic embryos. Thisis illustrated with two different antisense constructs in two differenttypes of experiment that were constructed following the protocols setforth in the PCT Publication Nos. WO 93/11245 and WO 94/11516. Liquidculture globular embryos were transformed with a chimeric genecomprising a soybean microsomal Δ¹⁵ desaturase as described in PCTPublication No. WO 93/11245 which was published on Jun. 10, 1993, thedisclosure of which is hereby incorporated by reference (experiment 1)or a soybean microsomal Δ¹² desaturase as described in PCT PublicationNo. WO 94/11516 which was published on May 26, 1994, the disclosure ofwhich is hereby incorporated by reference (experiment 2). Both geneconstructs were introduced in antisense orientation under the control ofa seed-specific promoter (β-conglycinin promoter) and gave rise tomature embryos. The fatty acid content of mature somatic embryos fromlines transformed with vector only (control) and the vector containingthe antisense chimeric genes as well as of seeds of plants regeneratedfrom them was determined.

One set of embryos from each line was analyzed for fatty acid contentand another set of embryos from that same line was regenerated intoplants. Fatty acid analysis of single embryos was determined either bydirect trans-esterification of individual seeds in 0.5 mL of methanolicH₂SO₄ (2.5%) or by hexane extraction of bulk seed samples followed bytrans-esterification of an aliquot in 0.8 mL of 1% sodium methoxide inmethanol. Fatty acid methyl esters were extracted from the methanolicsolutions into hexane after the addition of an equal volume of water. Inall cases, if there was a reduced 18:3 content in a transgenic embryoline when compared to an untransformed control, then a correspondingreduction in 18:3 content was also observed in the segregating seeds ofthe plant derived from that transformed line (Table 2). TABLE 2 Percent18:3 Content Of Embryos and Seeds of Control and Δ¹⁵ Antisense ConstructTransgenic Soybean Lines Embryo Average Seed Average Transformant Line(SD, n = 40) (SD, n = 10) Control 12.1 (2.6)  8.9 (0.8) Δ¹⁵ antisense,line 1 5.6 (1.2) 4.3 (1.6) Δ¹⁵ antisense, line 2 8.9 (2.2) 2.5 (1.8) Δ¹⁵antisense, line 3 7.3 (1.1) 4.9 (1.9) Δ¹⁵ antisense, line 4 7.0 (1.9)2.4 (1.7) Δ¹⁵ antisense, line 5 8.5 (1.9) 4.5 (2.2) Δ¹⁵ antisense, line6 7.6 (1.6) 4.6 (1.6)*[Seeds which were segregating with wild-type phenotype and without acopy of the transgene are not included in these averages]

In addition, different lines containing the same antisense construct,were used for fatty acid analysis in somatic embryos and forregeneration into plants. About 55% of the transformed embryo linesshowed an increased 18:1 content when compared with control lines (Table3). Soybean seeds, of plants regenerated from different somatic embryolines containing the same antisense construct, had a similar frequency(53%) of high oleate transformants as the somatic embryos (Table 3). Onoccasion, an embryo line may be chimeric. That is, 10-70% of the embryosin a line may not contain the transgene. The remaining embryos that docontain the transgene, have been found in all cases to be clonal. Insuch a case, plants with both wild type and transgenic phenotypes may beregenerated from a single, transgenic line, even if most of the embryosanalyzed from that line had a transgenic phenotype. An example of thisis shown in Table 4, in which, of 5 plants regenerated from a singleembryo line, 3 have a high oleic phenotype and two were wild type. Inmost cases, all the plants regenerated from a single transgenic linewill have seeds containing the transgene. Thus, it was concluded that analtered fatty acid phenotype observed in a transgenic, mature somaticembryo line is predictive of an altered fatty acid composition of seedsof plants derived from that line. TABLE 3 Oleate Levels in SomaticEmbryos and Seeds of Regenerated Soybeans Transformed With, or Without,Δ¹² Desaturase Antisense Construct # of # of Lines with Average* VectorLines High 18:1 %18:1 Somatic embryos: Control 19 0 12.0 Δ¹²antisense 2011 35.3 Seeds of regenerated plants: Control 6 0 18.2 Δ¹²antisense 17 944.4*average 18:1 of transgenics is the average of all embryos or seedstransformed with the Δ¹² antisense construct in which at least oneembryo or seed from that line had an 18:1 content greater than 2standard deviations from the control value (12.0 in embryos, 18.2 inseeds). The control average is the average of embryos or seeds which donot contain any transgenic DNA but have been treated in an identicalmanner to the transgenics.

TABLE 4 Analysis of Seeds From Five Independent Plants Segregating FromPlant Line 4 Plant # Average seed 18:1% Highest seed 18:1% 1 18.0 26.3 233.6 72.1 7 13.6 21.2 9 32.9 57.3 11 24.5 41.7Mean of 15-20 seeds from 5 different plants regenerated from a singleembryo line. Only plants # 2, 9 and 11 have seeds with a high 18:1phenotype.

Example 4 Analysis of Nucleic Acid Sequences

Nucleic acid sequences comprising the target regions or thecomplementary regions are analyzed by conducting BLAST (Basic LocalAlignment Search Tool; Altschul et al (1993) J. Mol. Biol. 215:403-410;see also www.ncbi.nlm.nih.gov/BLAST/) searches for similarity tosequences contained in the BLAST “nr” database (comprising allnon-redundant GenBank CDS translations, sequences derived from the3-dimensional structure Brookhaven Protein Data Bank, the last majorrelease of the SWISS-PROT protein sequence database, EMBL, and DDBJdatabases). The nucleic sequences are analyzed for similarity to allpublicly available DNA sequences contained in the “nr” database usingthe BLASTN algorithm provided by the National Center for BiotechnologyInformation (NCBI). The DNA sequences can also be translated in allreading frames and compared for similarity to all publicly availableprotein sequences contained in the “nr” database using the BLASTXalgorithm (Gish and States (1993) Nat. Genet. 3:266-272) provided by theNCBI. For convenience, the P-value (probability) of observing a match ofa cDNA sequence to a sequence contained in the searched databases merelyby chance as calculated by BLAST are reported herein as “pLog” values,which represent the negative of the logarithm of the reported P-value.Accordingly, the greater the pLog value, the greater the likelihood thatthe cDNA sequence and the BLAST “hit” represent homologous proteins.

Example 5 A Comparison of Reduced Fad2 Expression Using Antisense vs.“Classical” Co-Suppression vs. “Complementary Region” Co-Suppression

The following are some comparisons of antisense, “classical”co-suppression, and “complementary region” co-suppression (CRC) insimilar experiments involving a soybean fatty acid desaturase (Fad).Fad2-1 is a gene locus encoding a Δ-12 desaturase from soybean thatintroduces a double bond into the oleic acid side-chain to form apolyunsaturated fatty acid. Reduction in the expression of Fad2-1results in the accumulation of oleic acid (18:1, or an 18 carbon fattyacid tail with a single double bond) and a corresponding decrease inpolyunsaturated fatty acid content.

The antisense constructs have all, or a portion, of the Fad2-1 codingregion in a reverse orientation behind a strong promoter. It is believedthat expression of the “antisense” RNA interferes with normaltranslation of the homologous endogenous gene via a hybridization event.The “classical” co-suppression construct have all, or a portion, ofFad2-1 in the normal sense orientation behind a strong promoter. It isbelieved that the expression of the “co-suppressing” RNA activates anuncharacterized mechanism that results in the partial, or total,elimination of the introduced RNA, as well as all RNAs havingsubstantially similar sequences. The CRC construct used contains aportion of the Fad2-1 coding region (300 bp) duplicated in the reversecomplement orientation, forming a complementary region specific forFad2-1.

The plasmids used in these experiments were made using standard cloningmethods well known to those skilled in the art (Sambrook et al (1989)Molecular Cloning, CSHL Press, New York). A starting plasmid pKS18HH(U.S. Pat. No. 5,846,784 the contents of which are hereby incorporatedby reference) contains a hygromycin B phosphotransferase (HPT) obtainedfrom E. coli strain W677 under the control of a T7 promoter and the 35Scouliflower mosaic virus promoter. Plasmid pKS18HH thus contains the T7promoter/HPT/T7 terminator cassette for expression of the HPT enzyme incertain strains of E. coli, such as NovaBlue (DE3) [from Novagen], thatare lysogenic for lambda DE3 (which carries the T7 RNA Polymerase geneunder lacV5 control). Plasmid pKS18HH also contains the 35S/HPT/NOScassette for constitutive expression of the HPT enzyme in plants, suchas soybean. These two expression systems allow selection for growth inthe presence of hygromycin to be used as a means of identifying cellsthat contain the plasmid in both bacterial and plant systems. pKS18HHalso contains three unique restiction endonuclease sites suitable forthe cloning other chimeric genes into this vector. Plasmid ZBL100 (PCTApplication No. WO 00/11176 published on Mar. 2, 2000) is a derivativeof pKS18HH with a reduced NOS 3′ terminator. Plasmid pKS67 is a ZBL100derivative with the insertion of a beta-conglycinin promoter, in frontof a NotI cloning site, followed by a phaseolin 3′ terminator (describedin PCT Application No. WO 94/11516, published on May 26, 1994). PKS91 isa derivative of pKS67 with a polymerase chain reaction (PCR) hairpinfragment of the soybean Fad2 gene inserted into the Not I site. Theprimers used in PCR reactions with soybean Fad2-1 DNA as follows (allsequences are 5′ to 3′): PCR(A) GAATTCGCGGCCGCATGGGAGGTAGAGGTC SEQ IDNO:1 GGAAAACCATGCAACCCATTGGTACTTGCT SEQ ID NO:2 PCR(B)AGCAAGTACCAATGGGTTGCATGGTTTTCC SEQ ID NO:3AGCAAGTACCAATGGATACTTGTTCCTGTA SEQ ID NO:4 PCR(A/AS)TACAGGAACAAGTATCCATTGGTACTTGCT SEQ ID NO:5GAATTCGCGGCCGCATGGGAGGTAGAGGTC SEQ ID NO:6

The products of the three reactions (A)+(B)+(A/AS) are ligated together,digested with the restriction enzyme Not I, and the 1.3 kb fragment iscloned into the Not I site of KS67. The plasmid pKS91 was used in theexperiments presented in this section. The 2.5 kb plasmid pKS17 containspSP72 (obtained from Promega Biosystems) and the T7 promoter/HPT/T7 3′terminator region, and is the original vector into which the 3.2 kbBamHI-SalI fragment containing the 35S/HPT/NOS cassette was cloned toform pKS18HH. The plasmid pKS102 is a pKS17 derivative that is digestedwith XhoI and SalI, treated with mung-bean nuclease to generate bluntends, and ligated to insert the following linker:GGCGCGCCAAGCTTGGATCCGTCGACGGCGCGCC SEQ ID NO:7

The plasmid pKS83 has the 2.3 kb BamHI fragment of ML70 containing theKti3 promoter/NotI/Kti3 3′ terminator region (described in PCTApplication No. WO 94/11516, published on May 26, 1994) ligated into theBamHI site of pKS17. The plasmid pKS103 is a derivative of pKS83 withthe 1.3 kb NotI fragment of pKS91 (containing the Fad2 complementarysequence) ligated into the NotI site.

In order to have comparable numbers of antisense lines to compare to themore numerous co-suppression constructs, it was necessary to includeantisense experiments in which Fad 6 was co-bombarded with Fad 2-1. Fad6is a gene encoding Δ-12 desaturase found in plastids (as opposed to Fad2which is found in the microsomal compartment). It was believed thatsuppression of Fad2 and Fad6 simultaneously might give a stronger, ordifferent, phenotype than Fad2 suppression alone. However, it has sincebeen determined that Fad6 does not produce a phenotype, therefore thephenotypes obtained from antisense experiments with both Fad2 and Fad6only reflect changes in Fad2-1 content.

Control embryos (286 individuals) had an average 18:1 content of 9% witha standard deviation (SD) of 6.2% (actual range 4-22%). Thus, an oleicacid content of 25% was chosen to represent a positive reduction inFad2-1 which results in increased 18:1 that is more than 2 SDs from themean, and higher than the highest control value seen. If a line has atleast 1 embryo with an 18:1 content of 25% or more, it is counted as anantisense or a cosuppression event. Two experiments were combined togenerate about 30 lines. TABLE 5 Positive Transformed Lines With ReducedFad2-1 Expression Co- Antisense suppression CRC Fad2-1 lines with >25%18:1 9 out of 31 9 out of 28 31 out of 33 content Percent total 29% 32%94%

Another point to consider when analyzing transgenic plants with reducedexpression due to antisense or co-suppression is chimerism. In theantisense and cosuppression experiments above the positive events linesdetected may have only contained a single embryo out of ten withincreased oleic acid content. Since all of these experiments had 10embryos per line analyzed it is possible to graphically representchimerism data by plotting actual embryo numbers against oleic content(greater or less than 25% which would be indicative of a tranformantreduced in Δ-12 expression). Therefore, if a line has little or nochimerism then all of its embryos will have a suppressed phenotype asopposed to being wild types. The data appear to be quite convincing thatCRC (the grey box) transformants give consistently higher oleic acidcontents with less chirmeric events:

Another issue is the efficiency with which a line exhibits the reducedexpression phenotype. The results from the experiments here and inExample 6 confirm that the constructs containing the complementaryregions in proximity to the target sequence were more effective atproducing very high 18:1 content in embryos than either antisense or“classical” co-suppression (i.e. as opposed to complementary regioncontaining co-suppression, CRC). The level of suppression achieved in anexperiment is reflected in the corresponding increase of oleic acidcontent in the plants. The higher the average 18:1 content, the greaterthe degree of suppression. The complementary region containingconstructs had oleic acid contents of 50% which is over 5 SDs from thecontrol mean. TABLE 6 Positive Transformed Lines With High EfficiencyReduction in Fad2-1 Expression Co- Antisense suppression CRC Fad2-1lines with >50% 18:1 1 out of 31 3 out of 28 29 out of 33 contentPercent total 3% 11% 88%

It appears that CRC is the most efficient and effective way of producinghigh 18:1 content in embryos with reduced Fad2-1 content. As was shownin Example 3 there is a phenotypic correlation between embryo oleic acidcontent and seed oleic acid content in transgenic plant. Thusexperiments yielding embryonic lines with greater than 51% are mostdesirable since they appear to guarantee a seed oleic of greater than80%:

It is noted that most positive seed lines detected are close to (orgreater than) 80% oleic. Those few that aren't appear to be derived fromembryo lines with a maximum oleic content ranging from 30% to 50%. Todate no lines having a positive phenotype that had maximum embryocontent of oleic acid less than 30%, and lines in the production systemwith 51% or more oleic acid content have always given rise to the bestseed phenotypes. Additionally, the top five embryo lines from production(all greater than 50% oleic) gave the best phenotype in seed (greaterthan 80%) and the bottom four embryo lines (all less than 50% oleic inembryos) all gave less than 80% oleic acid content in seed.

Example 6 Target and Complementary Sequences can Both Co-Suppress theirEndogenous Homologs

The inclusion of a complementary region into the target region of aco-suppression construct results in the improvement of efficiency anduniformity in the resultant transformants (Example 5). The next step isto test if more than one gene can be suppressed using this approach.Preliminary results using a 300 nucleotide complementary region fromFad2-1 surrounding a 600 nucleotide target from the soybean thioesterasegene results in the suppression of both genes. This result wasinteresting for two reasons. First the complementary region from Fad2was interrupted with thioesterase sequence, unlike the constructpresented in Example 5. Second, the non-complementary target sequence(thioesterase) was inhibited in all lines that exhibited Fad2 reductionof expression, implying that there was equal efficiency of target andcomplementary region reduction of expression.

To further test if any target sequence expression can be efficientlyrepressed with any complementary region a construct was made usingFad2-1 as the target in combination with a complementary region from thesoybean eceriferum3 (cer3) locus. Cer3 encodes one of 21 gene productsknown to be involved in wax biosynthesis in Arabidopsis thaliana(Hannoufa et al (1996) Plant J 10:459-67). The inhibition of a singlecer3 gene has no visible phenotype in soybean. Also, cer3 is involved ina biosynthetic pathway that has no known interactions with the fattyacid metabolic pathway containing Fad2 activity. The plasmid pKS100 is aderivative of pKS67. PCR reactions are run with the following primers(5′-3′ orientations) and cer3 DNA: PCR(A + B)GAATTCGCGGCCGCGGCACGAGATTTGAGG SEQ ID NO:8TTGCCCAATGTTTATGCATATGTAGAACTG SEQ ID NO:9 PCR(A/AS)CAGTTCTACATATGCATAAACATTGGGCAA SEQ ID NO:10GAATTCGCGGCCGCGGCACGAGATTTGAGG SEQ ID NO:11

The product of the two reactions are ligated together, digested withNotI, and cloned into the NotI site of pKS67. Next, the 516 bp ScaIfragment from soybean Fad2-1 is ligated into a FspI digested pKS100(within the Cer3 DNA, removing a small portion of the complementarysequence) to form pBS58. Restriction enzyme digestions were used toselect the plasmid containing the Fad2 fragment in the sense orantisense orientation. It is believed that Fad2 expression is reducedefficiently in all constructs tested in soybeans.

Example 7 Suppression in Soybean of Fad2 By ELVISLIVES ComplementaryRegion

Constructs have now been made which have “synthetic complementaryregions” (SCR). Since the Fad 2 CR/TE2 target suppressed both endogenousgenes in the one line examined, and since Cer3/Fad2 constructs suppressFad2, it was deduced that it may be possible to use any complementarysequence to reduce the expression of a target. In this example thetarget sequence is placed between complementary sequences that are notknown to be part of any biologically derived gene or genome (i.e.sequences that are “synthetic” or conjured up from the mind of theinventor). The target DNA would therefore be in the sense or antisenseorientation and the complementary RNA would be unrelated to any knownnucleic acid sequence. It is possible to design a standard “suppressionvector” into which pieces of any target gene for suppression could bedropped. The plasmids pKS106, pKS124, and pKS133 exemplify this. Oneskilled in the art will appreciate that all of the plasmid vectorscontain antibiotic selection genes such as, but not limited to,hygromycin phosphotransferase with promoters such as the T7 induciblepromoter.

pKS106 uses the beta-conglycinin promoter while the pKS124 and 133plasmids use the Kti promoter, both of these promoters exhibit strongtissue specific expression in the seeds of soybean. pKS106 uses a 3′termination region from the phaseolin gene, and pKS124 and 133 use a Kti3′ termination region. pKS106 and 124 have single copies of the 36nucleotide EagI-ELVISLIVES sequence surrounding a NotI site (the aminoacids given in parentheses are back-translated from the complementarystrand): SEQ ID NO:12. EagI   E   L   V   I   S   L   I   V   E   S    NotI CGGCCG GAG CTG GTCATC TCG CTC ATC GTC GAG TCG GCGGCCGC (S) (E) (V) (I) (L) (S) (I) (V) (L)(E) EagI CGA CTC GAC GAT GAG CGA GAT GAC CAG CTC CGGCCG

pKS133 has 2× copies of ELVISLIVES surrounding the NotI site: SEQ IDNO:13  EagI  E  L  V  I  S  L  I  V  E  S    EagI   E  L  V  I  Scggccggagctggtcatctcgctcatcgtcgagtcg gcggccg gagctggtcatctcg L  I  V  E  S    NotI   (S) (E (V) (I) (L) (S) (I) (V) (L) (E)   EagIctcatcgtcgagtcg gcggccgc cgactcgacgatgagcgagatgaccagctc cggccgc(S)(E)(V)(I)(L)(S)(I)(V)(L)(E)  EagI cgactcgacgatgagcgagatgaccagctccggccg

The idea is that the single EL linker (SCR) can be duplicated toincrease stem lengths in increments of approximately 40 nucleotides. Aseries of vectors will cover the SCR lengths between 40 bp and the 300bp. Various target gene lengths are also under evaluation. It isbelieved that certain combinations of target lengths and complementaryregion lengths will give optimum suppression of the target, althoughpreliminary results would indicate that the suppression phenomenon workswell over a wide range of sizes and sequences. It is also believed thatthe lengths and ratios providing optimum suppression may vary somewhatgiven different target sequences and/or complementary regions.

The plasmid pKS106 is made by putting the EagI fragment of ELVISLIVES(SEQ ID NO:12) into the NotI site of pKS67. The ELVISLIVES fragment ismade by PCR using two primers and no other DNA:5′-GAATTCCGGCCGGAGCTGGTCATCTCGCTCATCG SEQ ID NO:14TCGAGTCGGCGGCCGCCGACTCGACGATGAGCGAGAT GACCAGCTCCGGCCGGAATTC-3′5′-GAATTCCGGCCGGAG-3′ SEQ ID NO:15

The product of the PCR reaction is digested with EagI (5′-CGGCCG-3′) andthen ligated into NotI digested pKS67. The pKS111 is made by inserting a599 nucleotide fragment from the delta-12 desaturase gene (Fad2,nucleotides 399-997), in an antisense orientation into the NotI site ofpKS106. The Fad2 fragment is made by PCR using the following primers andFad2 DNA as a template: GAATTCGCGGCCGCTGAGTGATTGCTCACGAGT SEQ ID NO:16GAATTCGCGGCCGCTTAATCTCTGTCCATAGTT SEQ ID NO:17

The PCR product is digested with NotI (5′-GCGGCCGC-3′) and ligated intoNotI digested pKS106. The total length of complementary sequence is 47nucleotides (with the 8 nucleotides from the NotI site and 3 additionalflanking bases). Co-suppression of Fad2 results in a decrease in theproduction of polyunsaturated fatty acids, and a corresponding increasein the accumulation of oleic acid (18:1) in soybeans. (see Example 3above). Oleic acid concentrations in 18 of the 22 lines transformed withpKS111 were 2-5 times that found for the vector only controls,indicating co-suppression in 82% of the recovered transgenic plants. Itappears that the placement of a single SCR (ELVISLIVES or EL)surrounding a short segment of Fad2 (600 bp) is sufficient to giveco-suppression at efficiencies equal to the efficiencies achieved usingthe CRC constructs of Example 5. The term “ELVISLIVES” and “EL” are usedinterchangeably herein.

Additional plasmids can be used to test this example. For example,pKS121 contains the Kti3 promoter/NotI/Kti3 3′ terminator fragmentanalogous to pKS83 inserted into the BamHI-SalI digested pKS102. TheEagI digested ELVISLIVES cloning site made from SEQ ID NOs:14 and 15 isinserted into the NotI site of pKS121 to form pKS124. The Fad2 fragmentfrom pKS111 is ligated into NotI digested pKS124 to form pKS132. TheEagI digested EL PCR product can be ligated into NotI digested pKS124 toform the 2XEL pKS133. An additional 2XEL vector, pKS151, is similar topKS133 except for the addition of a second hygromycin phosphotransferasegene with a 35S-CaMV promoter. Any synthetic sequence, or naturallyoccurring sequence, can be used in an analogous manner. The addition ofthe 599 bp soybean Fad2 fragment from pKS111 into a NotI digested pKS133produces pKS136.

The efficiency of Fad2 suppression using 1XEL (pKS132) was compared toFad2 suppression using the 2XEL (pKS136) construct. Hygromycin resistantlines of soybean embryos were isolated from independent transformationexperiments with pKS132 and pKS136. Out of 98 lines containing pKS132,69% displayed the high oleic phenotype. Out of 54 lines containingpKS136, 70% displayed the high oleic acid phenotype. Thus, both 1X and2XEL constructs efficiently suppressed the Fad2 target gene.

Example 8 Length of the Fad2 Target Sequence Affects SuppressionEfficiency

The length of the target was tested to determine the effect on theefficiency of suppression in an EL construct. PCR reactions wereperformed using the primers shown in Table 7 to create 25, 50, 75, 150,300, and 600 fragments of Fad2 to place between 2XEL complementaryregions. The PCR products were cut with Not I and ligated intopBluescript and the sequence of the fragments was verified. Not Idigested fragments were then ligated into the NotI of pKS151. TABLE 7Primers for PCR of Soybean Fad2 Primer Sequence Length SEQ ID NO5′-GAATTCGCGGCCGCCCAATCTATTGGGTTCTC-3′ — 18 common 5′-end primerposition 363 in Fad2 sequence 5′-GAATTCGCGGCCGCAACCTTGGAGAACCCAAT-3 2519 3′-end primer for 25 bp fragment from 363-387 of Fad25′-GAATTCGCGGCCGCATCACCCACACACCAGTG-3′ 50 36 3′-end primer for 50 bpfragment from 363-412 of Fad2 5′-GAATTCGCGGCCGCGGCATGGTGACCACACTC-3′ 7520 3′-end primer for 75 bp fragment from 363-437 of Fad25′-GAATTCGCGGCCGCTGAGAAATAAGGGACTAA-3′ 150 21 3′-end primer for 150 bpfragment from 363-512 of Fad2 5′-GAATTCGCGGCCGCGAGTGTGACGAGAAGAGA-3′ 30022 3′-end primer for 300 bp fragment from 363-662 of Fad25′-GAATTCGCGGCCGCTTCTGATGAATCGTAATG-3′ 600 23 3′-end primer for 600 bpfragment from 363-962 of Fad2

TABLE 8 Effect of Target Length on Suppression by 2XEL Fad2 TargetLength # Lines Tested High Oleic 25 8 0% 50 8 0% 75 8 13% 150 8 13% 30029 34% 600 20 60%

The results in Table 8 show a clear correlation between target lengthand efficiency of suppression. The longest (600 bp) fragment of Fad2 isnearly twice as likely to be suppressed in the EL construct than a 300bp fragment, while 50 bp and shorter fragments are not effective.

Example 9 Multiple Target Sequences can be Suppressed by 2XEL

A construct was assembled to test whether multiple target sequences canbe used between EL complementary sequences to achieve simultaneoussuppression. A 969 bp fragment from a soybean delta-9 desaturase wasinserted into pKS136 next to the 599 bp Fad2 fragment to form pBS68.Both desaturase fragments were flanked by 2XEL complementary regions(2×EL-Fad2-Delta 9-2XEL the sequence of which is shown in SEQ ID NO:24).

Delta-9 desaturase catalyzes the double-bond at the 9-position of18-carbon fatty acids to form oleic acid (18:1) from stearic acid(18:0), analogous to the delta-12 Fad2 which catalyzes the 12-positiondouble bond that converts oleic acid to linoleic acid (18:2).Suppression of the unique Fad2 gene results in an accumulation of oleicacid at the expense of polyunsaturated fatty acids. Suppression ofdelta-9 desaturases results in an accumulation of stearic acid at theexpense of all unsaturated fatty acids. However, there are severaldelta-9 desaturases in soybean (at least three) so it is unclear how thesuppression of one member would affect oil composition. Transformationprotocols and oil composition analyses were performed as previouslyoutlined in Examples 1 and 3, respectively.

Transformation of soybean with pBS68 resulted in 113 hygromycinresistant lines. Of these 72 showed some oil phenotype (64%). Thephenotypes of the 72 suppressed lines were: 18 were high stearate, 23were high oleate, and 31 were both high oleate and high stearate.Therefore, multiple targets can be efficiently suppressed by a single ELconstruct.

Example 10 Suppression of Soybean Galactinol Synthase Genes inELVISLIVES Constructs

Raffinose saccharides are a group of D-galactose-containingoligosaccharide derivatives of sucrose that are widely distributed inplants. Raffinose saccharides are characterized by the general formula:[O-β-D-galactopyranosyl-(1→6)_(n)-α-glucopyranosyl-(1→2)-β-D-fructofuranosidewhere n=0 through n=4 are known respectively as sucrose, raffinose,stachyose, verbascose, and ajugose.

Although abundant in many species, raffinose saccharides are an obstacleto the efficient utilization of some economically-important cropspecies. Raffinose saccharides are not digested directly by animals,primarily because alpha-galactosidase is not present in the intestinalmucosa [Gitzelmann et al (1965) Pediatrics 36:231-236; Rutloff et al(1967) Nahrung 11:39-46]. However, microflora in the lower gut arereadily able to ferment the raffinose saccharides resulting in anacidification of the gut and production of carbon dioxide, methane andhydrogen gases [Murphy et al (1972) J Agr. Food Chem. 20:813-817;Cristofaro et al (1974) in Sugars in Nutrition, H. L. Sipple and K. W.McNutt, Eds. Academic Press, New York, Chap. 20, 313-335; Reddy et al(1980) J Food Science 45:1161-1164]. The resulting flatulence canseverely limit the use of leguminous plants in animal, particularlyhuman, diets. It is unfortunate that the presence of raffinosesaccharides restricts the use of legumes in human diets because many ofthese species are otherwise excellent sources of protein and solublefiber. Varieties of edible beans free of raffinose saccharides would bemore valuable for human and animal diets and would facilitate broaderaccess to the desirable nutritional qualities of edible leguminousplants.

The biosynthesis of raffinose saccharides has been well characterized[see Dey (1985) in Biochemistry of Storage Carbohydrates in GreenPlants, P. M. Dey and R. A. Dixon, Eds. Academic Press, London, pp.53-129]. The committed reaction of raffinose saccharide biosynthesisinvolves the synthesis of galactinol from UDP-galactose andmyo-inositol. The enzyme that catalyzes this reaction is galactinolsynthase (inositol 1-alpha-galactosyltransferase; EC 2.4.1.123).Synthesis of raffinose and higher homologues in the raffinose saccharidefamily from sucrose is thought to be catalyzed by distinctgalactosyltransferases (for example, raffinose synthase and stachyosesynthase). Studies in many species suggest that galactinol synthase isthe key enzyme controlling the flux of reduced carbon into thebiosynthesis of raffinose saccharides [Handley et al (1983) J. Amer.Soc. Hort. Sci. 108:600-605; Saravitz, et al (1987) Plant Physiol.83:185-189]. Altering the activity of galactinol synthase, either as aresult of overexpression or through antisense inhibition, would changethe amount of raffinose saccharides produced in a given tissue.

Related galactinol synthase genes already known in the art includesequences disclosed in U.S. Pat. Nos. 5,773,699 and 5,648,210, Kerr etal, “Nucleotide Sequences of Galactinol Synthase from Zucchini andSoybean” and Sprenger and Keller (2000) Plant J 21:249-258. Presumablyrelated sequences are also disclosed in PCT Publication No. WO 98/50553,Lightner, “Corn Glycogenin”. Two genes encoding soybean galactinolsynthases have been previously identified (SEQ ID NOs:30 and 32, withthe predicted translation products shown in SEQ ID NOs:31 and 33;presented in U.S. Pat No. U.S. Provisional Application No. 60/196,550,filed Apr. 11, 2000). Unlike the unique soybean Fad2 gene, it is knownthat there are multiple galactinol synthase genes in soybean. Becausethere are multiple genes encoding galactinol synthases, it is believedthat suppression of more than one gene may be required to detect aneffect on raffinose sugar levels.

A plasmid construct was assembled containing fragments of two galactinolsynthase soybean genes Gas1 (390 bp from 13-402 of SEQ ID NO:30) andGas2 (399 bp, from 129-527 of SEQ ID NO:32) cloned in the NotI site of a2XEL cassette. The promoter region was a late embryo promoter (Lea) fromsoybean. The Lea promoter (Lee et al (1992) Plant Physiol 100:2121-2122;Genbank Accession No. M97285) was amplified from genomic A2872 soybeanDNA with the following primers: SEQ ID NO:25 5′-ATT AAC CTC AAT TCT TCTAAG (position 25-45 of M97285) SEQ ID NO:26 5′-TTC AAA GAT CAA TTA TTTCC (position 995-1112 M97285)and a phaseolin 3′-end (amplified with primers shown in SEQ ID NOs:27and 28) was added. The entire Lea promoter-2XEL-Gas1-Gas2-2XEL-phaseolin 3′-end cassette was then cloned into theBamHI site of pKS136 to create the pKS149 vector (the sequence of thecomplete EL region of pKS149 is shown in SEQ ID NO:29). When introducedinto plants pKS136 will inhibit both Fad2 (controlled by the Ktipromoter) and Gas genes (controlled by the Lea promoter). Since the Ktipromoter is active in embryos, it is possible to screen the embryos forhigh oleic phenotype, as described in the previous examples. Of the 119lines isolated as hygromycin resistant 65% were found to have a higholeic phenotype.

These suppressed lines should also contain the Gas suppression cassette,allowing for the assay of raffinose sugars in the seedlings (Lea is notactive during the early embryo stage). Raffinose sugars (galactinol,raffinose, stachyose, etc.) can be detected using thin layerchromatography. Plant samples are extracted with hexane then dried. Thedried material is then resuspended in 80% methanol, incubated at roomtemperature for 1-2 hours, centrifuged, and 1-2 microliters of thesupernatant is spotted onto a TLC plate (Kieselgel 60 CF, from EMScientific, Gibbstown, N.J.; catalog no. 13749-6). The TLC is run inethylacetate:isopropanol:20% acetic acid (3:4:4) for 1-1.5 hours. Theair dried plates are sprayed with 2% sulfuric acid and heated until thecharred sugars are detected. As shown in FIG. 2 the two lines labeledGAS-EL show reduced levels of raffinose sugars (lowest band) whencompared to a control known to have very low raffinose sugars (Low 4).It is estimated that there is a 60% reduction of raffinose sugars inthese lines when compared to wild-type soybean.

Example 11 ELVISLIVES Constructs can be Used to Screen Essential PlantGenes

Acetolactate synthase (ALS), also known as acetohydroxyacid synthase(AHAS), catalyzes the first common step in the biosynthesis of thebranched chain amino acids isoleucine, leucine, and valine (Keeler etal, Plant Physiol 1993 102: 1009-18). Inhibition of native plant ALS byseveral classes of structurally unrelated herbicides includingsulfonylureas, imidazolinones, and triazolopyrimidines, is lethal (ChongC K, Choi J D Biochem Biophys Res Commun 2000 279:462-7). Hencesuppression of the gene encoding ALS in soybean should also be lethal.Thus, a well-validated herbicide target like ALS can inserted into ELvectors to test whether the transformation screening process can be usedto identify essential plant genes. If so, other essential plant genescould be screened in a high-throughput method to identify novelpotential herbicide targets. The term “essential plant genes” as usedherein refers to genes encoding a product that is required for normalplant growth, development, and/or viability. In addition to ALS,examples of essential plant genes would include, but not be limited to,rate-limiting enzymes in amino acid, nucleic acid, or lipidbiosynthesis. It is also believed that many genes with unknown functionmay be essential.

If a soy EL-ALS-EL construct is expressed during selection onhygromycin, very few events should be recovered, even though the HPTgene is present. If the EL-ALS-EL transcriptional unit is not expresseduntil late embryogenesis then recovery of transformation events shouldbe similar in number to events obtained with vector controls, containingonly the HPT gene. Constitutive expression of EL-ALS-EL can beaccomplished by using a 35S promoter (pKS161). Expression of EL-ALS-ELrestricted to late embryogenesis/germination can be accomplished withthe previously described LEA promoter (pKS163).

To make KS161 the EL linker (SEQ ID NO:12) was cloned into the NotI siteof pKS50 to produce pKS137 (a single EL complementary region with a 1 kb35S CaMV promoter and a 700 bp nos 3′-end on a plasmid with 2 HPT genesone with a T7 promoter and the second with a 35S promoter). A 208 bpHind III/EcoR I fragment from a soybean ALS gene (SEQ ID NO:35, fragmentis from position 891-1114) was then cloned into the Hind III/EcoR Isites of pKS137 to produce pKS161. To make pKS163 the EL linker (SEQ IDNO:12) was cloned into the NotI site of pKS127 to produce KS139 (asingle EL complementary region with the Lea promoter and the phaseolin3′-end from Example 10 on a plasmid with 2 HPT genes one with a T7promoter and the second with a 35S promoter). The 208 bp Hind III/EcoR Ifragment from soybean ALS gene (SEQ ID NO:35, the HindIII/EcoRI fragmentis from position 896-1103) was then cloned into the Hind III/EcoR Isites of KS139 to produce KS163.

KS161 and KS163 were transformed into 821 tissue (Example 1). Thetransformation efficiency for this tissue is normally in the range of200-500 clones/gram of tissue. The results of two separatetransformation experiments with KS161 and KS163, 4 weeks afterbombardment and transfer to hygromycin-containing medium are: Expt. 1KS161 (35S ALS EL)  16 clones/gram tissue KS163 (LEA ALS EL) 247clones/gram tissue Expt. 2 KS161 (35S ALS EL)  43 clones/gram tissueKS163 (LEA ALS EL) 467 clones/gram tissue

In both experiments the 35S EL-ALS vector resulted in a >90% decrease inclone numbers, presumably because of suppression of the endogenous ALSgene throughout embryo formation stages. Therefore, the difference inclone numbers obtained for a novel gene fragment inserted into a 35S-ELconstruct (KS137) and a LEA-EL construct (KS139) can be used as ameasure of whether the corresponding endogenous gene is essential ornot, and thus whether or not it is a potential herbicide target. Theeffect of an unknown gene fragment on transformation efficiency can bemeasured within a few weeks of particle bombardment and thus this is arapid means of identifying new herbicide target candidates. A typicalscreen consists of bombarding tissue with KS137 and KS139 asempty-vector controls, KS161 as a positive (ALS) control and variousgene fragments, amplified by PCR to contain Hind III and EcoR I sites,cloned into the HindIII/EcoRI sites of KS137 and KS139.

The improved frequency of suppression achieved with the EL constructsallows for the possibility of a reliable screening method. A significantpercentage of the hygromycin recovered transformation events must besuppressed by the target sequence contained within pKS137 or pKS139 inorder for there to be a statistically definitive difference between thetwo experiments. The term “high degree of frequency” as used herein,with respect to the suppression efficiency, refers to the percentage oftransformed lines that exhibit the target suppressed phenotype. Highfrequency percentages are expected to be in a range of at least 15-95%and any integer percentage found within the range. Preferred embodimentswould include at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, and 95%.

Example 12 Suppression of Cellulose Synthase in Maize Using ELConstructs

Cellulose synthase genes encode a family of proteins involved incellulose formation in plants (Pear, et al, Proc. Natl. Acad. Sci. (USA)93, 12637-12642; Saxena, et al, (1990), Plant Molecular Biology 15,673-684). Several maize genes encoding cellulose synthases (cesA) havebeen recently cloned and characterized (PCT Publication No. WO 00/09706,published on Feb. 24, 2000). Fragments from four of these genes, cesA1,cesA4, cesA5, and cesA8, were used to test whether 1XEL could direct thesuppression of these genes in maize.

One kb fragments from the 5′-end of the cDNA clones (including 5′-UTRand ORF sequences) for cesA1, cesA4, cesA5, and cesA8 were each clonedseparately into the internal NotI site of 1XEL (SEQ ID NO:12)constructs. Each of these “EL-cesA-EL” cassettes was inserted into aplasmid containing a f3.7 promoter (a weak constitutive promoterexhibiting some preference for stalk-specific expression), a proteinaseinhibitor 3′-end (pinII from potato, An et al (1989) Plant Cell 1:115-122), and a 35S:BAR:pinII selection marker. A control plasmidcontaining an IN2 promoter driving a GUS gene with a pinII 3′-end wasalso made.

Results from maize transformation experiments with each of theconstructs are shown in Table 9. Twenty-five lines were isolated foreach of the four cesA gene constructs and 18 lines were isolated for thecontrol. The height of the plants and stalk diameter were on averagesmaller in the lines containing the suppression constructs than in thecontrol. Ear heights were shorter in the cesA1 and cesA5 containinglines. The average cellulose percentage of total dry matter is normally46% in control plants. All of the cesA constructs had lines that werebelow 46% cellulose with cesA1>cesA5>cesA8>cesA4. The lines thatexhibited low cellulose percentages were tested by DNA Southern blotanalyses to determine which contained a single-copy transgene insertion.All had at least one line that had both low cellulose and a singletransgene. TABLE 9 Summary of CesA Suppression By EL Constructs heightEar Stalk Cellulose Single Construct (cm) (cm) (mm) <46% transgeneControl 171 44 16 CesA1 150 40 14 9 3 CesA4 164 46 13 3 2 CesA5 148 4113 7 2 CesA8 166 46 15 5 1

These results show that cellulose levels are altered in plantscontaining cesA gene fragments contained within 1XEL constructs. This isinterpreted as meaning that cesA suppression in maize has a detectablephenotype, and that EL-controlled suppression is active in maize. Itshould be noted that the f3.7 promoter is a weak promoter compared toothers used in this application (35S-CaMV, Kti, etc.) and that cesA is alarge multigene family. These factors may have an effect on thefrequency and/or extent of suppression.

Example 13 Transient Suppression of GFP in Maize Using GUS ComplementaryRegion

All expression cassettes used in this example comprise a maize ubiquitinpromoter (nt 1-899), a maize ubiquitin 5′ untranslated leader sequence(nt 900-982) and a maize ubiquitin intron 1 (nt 983-1992). In plasmidPHP7921 the coding sequence (nt 2015-2731) is GFP (green fluorescentprotein) with codons optimized for expression in maize. In plasmidPHP3953 the coding sequence (nt 2013-3821) is GUS. Both cassettesinclude the polyadenylation signal sequences from the proteinaseinhibitor II gene of S. tuberosum (PINII TERM, nt 2737-3047 in PHP7921and nt 3883-4192 in PHP3953).

Standard recombinant DNA methodologies well known to those skilled inthe art were used throughout the construction of the expressioncassettes for this work. Orientations of fragment insertions and thefinal structures of the plasmid constructs were determined usingstandard agarose gel analysis and/or sequencing of the plasmids.

Plasmid PHP7921 was used to create a complementary region (CR) of asmall portion of the GFP coding sequence as follows: plasmid DNA wasdigested with XhoI and treated with the Klenow fragment of DNApolymerase I to release a 244 bp blunt-ended fragment representing nt2436-2675 near the 3′ end of the GFP coding sequence. This fragment wasthen inserted back into PHP7921 at the HpaI site (nt 2735) justdownstream of the stop codon of GFP. A recombinant plasmid wasidentified that had the inserted fragment in the reverse orientationrelative to the original sequence. This plasmid was designated PHP16391.

Expression cassettes for GUS containing a heterologous GFP-CR wereconstructed as follows: the entire GFP-CR of PHP16391 was isolated as aBsrGI fragment (nt 2464-2947, 483 bp). This fragment comprises sequencescapable of forming a CR with a 214 bp stem and a 55 nt loop. Thefragment was rendered blunt-ended as above using Klenow and insertedinto the GUS expression cassette of PHP3953 at three different sites.Plasmid PHP16561 has the GFP-CR inserted in the BamHI site (filled in)of PHP3953 (nt 2006), just 5′ to the start codon. Plasmid PHP16562 hasthe GFP-CR inserted in the PacI site (T4 polymerase-treated to renderblunt) at nt 3919 of PHP3953 just 3′ to the stop codon. Similarly,plasmid PHP16563 has the GFP-CR inserted in the SnaBI site at nt 2398 ofPHP3953 within the GUS coding sequence.

High type II callus was maintained by subculturing onto fresh 560P (N6salts, Erikkson's vitamins, 0,69 g/l proline, 2 mg/l 2,4-D and 3%sucrose) medium every two weeks. Healthy callus was extruded through a0.77 mm² nylon mesh, weighed, and resuspended in MS culture medium with2 mg/l 2,4-D at a density of 3 grams of tissue/40 ml medium. Cells wereuniformly suspended by pipetting the solution up-and-down through alarge-bore pipette, and 4 ml aliquots (300 mg) were then collected onglass filter papers using a vacuum apparatus. These filters, eachcontaining approximately 300 mg of cells, were then placed on 560Pmedium and cultured in the dark at 26° C. After 2-4 days the filterswere removed from the culture medium and excess liquid was removed usinga vacuum apparatus. DNA was then delivered into the cells using a DuPontBiolistics particle gun using a standard Hi-II bombardmenttransformation protocol (Songstad D. D. et al, In Vitro Cell Dev. Biol.Plant 32:179-183, 1996) modified by using 1 um gold particles.Immediately after bombardment the filters were returned to 560P culturemedium and cultured in the dark at 26° C. All DNA's were combined inequal ratios to obtain a final concentration of 1 ug of totalDNA/particle preparation (0.33 ug of each DNA combined in eachpreparative tube was used for 6 shots). The experiment was shot asfollows: From each treatment 10 plates were bombarded (5 from each oftwo DNA preps). Treatment DNA's #1 Control PHP3953 (GUS) +PHP10256(rLuciferase) + PHP7921 (GFP) #2 GUS w/5′CRC PHP3953 (GUS) +PHP10256 (rLuciferase) + PHP16561(CR-GUS) #3 GUS w 3′ CRC PHP3953(GUS) + PHP10256 (rLuciferase) + PHP16562(GUS-CR) #4 GUS w/CRC in cdsPHP3953 (GUS) + PHP10256 (rLuciferase) + PHP16563(GU-CR-S)

For analysis, the plates within a treatment were grouped into 5 pairs(each pair containing plates shot with different DNA preparations forthe same plasmid treatment). Two days after bombardment, all the tissuefrom the two paired plates was combined and resuspended in 5 ml ofculture medium. After mixing with a wide-bore pipette, a 1 ml aliquotwas transferred into a 1.5 ml Eppendorf tube. The cells were centrifugedat 1000 RPMs for 2 minutes in a microfuge and the supernatant (culturemedium) decanted. To each tube, 150 ul of “Promega Luciferase PassiveLysis Buffer” was added, temperature-equilibrated on ice, and the cellswere broken apart using a hand-drill powered Kontes-tube plastic pestil(extraction and subsequent luciferase assays followed Promega'sDual-Luciferase Reporter Assay protocol (Technical Bulletin #TM040). Thecell debris was pelleted by centrifugation in the Microfuge at 3000 RPMfor 3 minutes and the supernatant was pipetted off. Aliquots of thisextract were used for the fluorometric quantitation of both GUS andluciferase (50 and 20 ul, respectively). GUS enzyme activity wasdetermined as a rate measurement between 10 and 40 minutes after addingsubstrates, and data was expressed as pmol MU/min/ml/extract (slope).Fluorometric GUS assays were performed on a LabSystems FLUOROSKAN AscentFL according to the protocol of Rao and Flynn (Biotechniques 19908:38-40. Fluorometric analysis of luciferase activity collected using anAnalytical Luminescence Laboratory Monolight 2010, following themanufacturer's instructions and the Promega protocol. Assessing bothmarkers for each replicate provided an internal control (luciferase)against which relative GUS activity could be rated. Thus, data wasplotted as the ratio of GUS/Luciferase units (because the absolutefluorometric units for Renilla luciferase were so high, all the rawvalues for luciferase activity were divided by 10 before using tonormalize GUS activity). TABLE 10 Complementary Regions of GFP ReduceTarget GUS Activity in a 3′ (#3) and Internal Orientation (#4) Pmol.Treatment MU/min/ml extract slope/rLuci Ave. Standard deviation #1 74.1427.66 #2 82.05 20.99 #3 20.22 17.87 #4 9.67 3.02

Repeat Expt Pmol. MU/min/ml extract Treatment slope/rLuci Ave. Standarddeviation #1 86.93 6.94 #2 77.3 22.62 #3 20.03 7.32 #4 11.2 2.32

It appears from the results (see Table 10) of these transient expressionexperiments that the placement of a complementary region 5′ to a targetdoes not reduce the expression of the target gene. However, it isbelieved that under optimized conditions, or in a stable transformationexperiment, placement of a complementary region 5′ to a target issufficient to reduce expression of the target.

Example 14 Suppression of Maize PDS by a Modified Soybean ComplementaryRegion

An additional suppression construct was created using a 205 bpHindIII-BstEII fragment from the soybean Kti promoter as thecomplementary region surrounding a multiple cloning site. Two copies ofthe Kti fragment were ligated in an inverted repeat arrangement andsubsequently modified by PCR to remove inconvenient restriction sitesand add cloning sites at both ends and in the region between the twocomplementary sequences to form the SHH3 cassette (see SEQ ID NO:34).The resulting plasmid (PHP17962) was used as a source of the SHH3sequence. The Kti sequence is not normally found in the maize genome,therefore no suppression of endogenous maize genes is expected from theSHH3 region alone. However, when a portion of an endogenous targetsequence is inserted into the cloning sites between the complementaryKti sequences, the homologous endogenous gene transcript should beaffected.

To test the utility of the SHH3 in silencing an endogenous gene, a 1385bp NheI fragment representing about 80% of the coding sequence of thephytoene desaturase gene (PDS-1) of Z. mays (Pioneer EST cnlcz91R,Genbank Accession No. L39266) was treated with Klenow enzyme aspreviously described to render the ends blunt and then ligated into theEcoRV site of SHH3 to generate PHP17894. The SHH3-PDS fragment was thenmoved as a 1865 bp HpaI fragment into an intermediate vector constructto place it under the control of the ubiquitin promoter:ubiquitin intron1 (U.S. Pat. Nos. 5,510,474 and 5,614,399) with polyadenylation signalsprovided by the pinII terminator (An et al (1989) Plant Cell 1:115-122). The resulting plant transcription unit was moved into a binaryvector (PHP15578) containing a CaMV35S-bialaphos selectable markerelement to generate PHP17914. This construct was electroporated intocompetent cells of Agrobacterium tumefaciens strain LBA4404 carrying thesuperbinary plasmid pSB1 ((Ishida et al (1996) Nature Biotech14:745-750). This process generates a cointegrate plasmid comprising thecombined sequences of PHP17914 and pSB1. This cointegrate plasmid,designated PHP17939, was used to transform immature embryos of Z. maysas follows.

Transformation of Maize Mediated by Agrobacterium

Freshly isolated immature embryos of maize, about 10 days afterpollination (DAP), were incubated with the Agrobacterium. The preferredgenotype for transformation is the highly transformable genotype Hi-II(Armstrong (1991) Maize Gen Coop Newsletter 65:92-93). An F₁ hybridcreated by crossing with an Hi-II with an elite inbred may also be used.After Agrobacterium treatment of immature embryos, the embryos werecultured on medium containing toxic levels of herbicide. Only thosecells which receive the herbicide-resistance gene, and the linkedgene(s), grow on selective medium. Transgenic events so selected arepropagated and regenerated to whole plants, produce seed, and transmittransgenes to progeny.

Preparation of Agrobacterium

The engineered Agrobacterium tumefaciens LBA4404 was constructed as perU.S. Pat. No. 5,591,616 to contain the PDS gene suppressed by thecomplementary region shown in SEQ ID NO:34 and a selectable marker gene.Typically either BAR (D'Halluin et al (1992) Methods Enzymol.216:415-426) or PAT (Wohlleben et al (1988) Gene 70:25-37) may be usedas a selectable marker.

To use the engineered construct in plant transformation, a master plateof single bacterial colonies was first prepared by inoculating thebacteria on minimal AB medium [minimal AB medium contains the followingingredients: 850.000 ml of deionized water; 50.000 ml of stock solution800A; 9 g of Phytagar which is added after Q.S. to volume; 50.000 ml ofstock solution 800B #; 5.000 g of glucose #; and 2.000 ml ofspectinomycin 50/mg/ml stock #. Directions are: dissolve ingredients inpolished deionized water in sequence; Q.S. to volume with polisheddeionized water less 100 ml per liter; sterilize and cool to 60° C.Ingredients designated with a # are added after sterilizing and coolingto temperature. Stock solution 800A contains the following ingredients:950.000 ml of deionized water; 60.000 g of potassium phosphate dibasicK2HPO4; and 20.000 g of sodium phos. monobasic, hydrous. Directions are:dissolve ingredients in polished deionized water in sequence; adjust pHto 7.0 with potassium hydroxide; Q.S. to volume with polished deionizedwater after adjusting pH; and sterilize and cool to 60° C. Stocksolution 800B contains the following ingredients: 950.000 ml ofdeionized water; 20.000 g of ammonium chloride; 6.000 g of magnesiumsulfate 7-H2O, MgSO4, 7H2O; 3.000 g of potassium chloride; 0.200 g ofcalcium chloride (anhydrate); and 0.050 g of ferrous sulfate 7-hydrate.Directions are: dissolve ingredients in polished deionized water insequence; Q.S. to volume with polished deionized water; and sterilizeand cool to 60° C.] and then incubating the bacteria plate inverted at28° C. in darkness for about 3 days. A working plate was then preparedby selecting a single colony from the plate of minimal A medium [minimalA medium contains the following ingredients: 950.000 ml of deionizedwater; 10.500 g of potassium phosphate dibasic K2HPO4; 4.500 g ofpotassium phosphate monobasic KH2PO4; 1.000 g of ammonium sulfate; 0.500g of sodium citrate dihydrate; 10.000 ml of sucrose 20% solution #; and1.000 ml of 1M magnesium sulfate #. Directions are: dissolve ingredientsin polished deionized water in sequence; Q.S. to volume with deionizedwater; sterilize and cool to 60° C. Ingredients designated with a # areadded after sterilizing and cooling to temperature] and streaking itacross a plate of YP medium [minimal YP medium contains the followingingredients: 950.000 ml of deionized water; 5.000 g of yeast extract(Difco); 10.000 g of peptone (Difco); 5.000 g of sodium chloride; 15.000g of bacto-agar, which is added after Q.S. to volume; and 1.000 ml ofspectinomycin 50 mg/ml stock #. Directions are: dissolve ingredients inpolished deionized water in sequence; adjust pH to 6.8 with potassiumhydroxide; Q.S. to volume with polished deionized water after adjustingpH; sterilize and cool to 60° C. Ingredients designated with a # areadded after sterilizing and cooling to temperature]. The YP-mediumbacterial plate was then incubated inverted at 28° C. in darkness for1-2 days.

Agrobacterium for plant transfection and co-cultivation was prepared 1day prior to transformation. Into 30 ml of minimal A medium in a flaskwas placed 50 μg/ml spectinomycin, 100 μM acetosyringone, and about a ⅛loopful of Agrobacterium from a 1 to 2-day-old working plate. TheAgrobacterium was then grown at 28° C. at 200 rpm in darkness overnight(about 14 hours). In mid-log phase, the Agrobacterium was harvested andresuspended at 3 to 5×10⁸ CFU/ml in 561Q medium+100 μM acetosyringoneusing standard microbial techniques and standard curves.

Immature Embryo Preparation

Nine to ten days after controlled pollination of a corn plant,developing immature embryos are opaque and 1-1.5 mm long and are theappropriate size for Agro-infection. The husked ears were sterilized in50% commercial bleach and 1 drop Tween for 30 minutes, and then rinsedtwice with sterile water. The immature embryos were aseptically removedfrom the caryopsis and placed into 2 ml of sterile holding solutioncomprising of medium 561Q+100 μM acetosyringone [medium 561 Q containsthe following ingredients: 950.000 ml of D-I Water, Filtered; 4.000 g ofChu (N6) Basal Salts (Sigma C-1416); 1.000 ml of Eriksson's Vitamin Mix(1000× Sigma-1511); 1.250 ml of Thiamine.HCL.4 mg/ml; 3.000 ml of 2,4-D0.5 mg/ml (No. 2A); 0.690 g of L-proline; 68.500 g of Sucrose; and36.000 g of Glucose. Directions are: dissolve ingredients in polisheddeionized water in sequence; adjust pH to 5.2 w/KOH; Q.S. to volume withpolished deionized water after adjusting pH; and filter sterilize (donot autoclave)].

Agrobacterium Infection and Co-Cultivation of Embryos

Holding solution was decanted from excised immature embryos and replacedwith prepared Agrobacterium. Following gentle mixing and incubation forabout 5 minutes, the Agrobacterium was decanted from the immatureembryos. Immature embryos were then moved to a plate of 562P medium[medium 562 P contains the following ingredients: 950.000 ml of D-IWater, Filtered; 4.000 g of Chu (N6) Basal Salts (Sigma C-1416); 1.000ml of Eriksson's Vitamin Mix (1000× Sigma-1511); 1.250 ml ofThiamine.HCL.4 mg/ml; 4.000 ml of 2,4-D 0.5 mg/ml; 0.690 g of L-proline;30.000 g of Sucrose; 3.000 g of Gelrite, which is added after Q.S. tovolume; 0.425 ml of Silver Nitrate 2 mg/ml #; and 1.000 ml of AcetoSyringone 100 mM #. Directions are: dissolve ingredients in polisheddeionized water in sequence; adjust pH to 5.8 w/KOH; Q.S. to volume withpolished deionized water after adjusting pH; and sterilize and cool to60° C. Ingredients designated with a # are added after sterilizing andcooling to temperature], scutellum surface upwards, and incubated at 20°C. for 3 days in darkness followed by incubation at 28° C. for 3 days indarkness on medium 562P+100 mg/ml carbenecillin (see U.S. Pat. No.5,981,840).

Selection of Transgenic Events

Following incubation, the immature embryos were transferred to 563Omedium [medium 563 O contains the following ingredients: 950.000 ml ofD-I Water, Filtered; 4.000 g of Chu (N6) Basal Salts (Sigma C-1416);1.000 ml of Eriksson's Vitamin Mix (1000× Sigma-1511); 1.250 ml ofThiamine.HCL.4 mg/ml; 30.000 g of Sucrose; 3.000 ml of 2,4-D 0.5 mg/ml(No. 2A); 0.690 g of L-proline; 0.500 g of Mes Buffer; 8.000 g of Agar(Sigma Δ-7049, Purified), which is added after Q.S. to volume; 0.425 mlof Silver Nitrate 2 mg/ml #; 3.000 ml of Bialaphos 1 mg/ml #; and 2.000ml of Agribio Carbenicillin 50 mg/ml #. Directions are: dissolveingredients in polished deionized water in sequence; adjust to pH 5.8w/koh; Q.S. to volume with polished deionized water after adjusting pH;sterilize and cool to 60° C. Ingredients designated with a # are addedafter sterilizing and cooling to temperature] for selection of events.The transforming DNA possesses a herbicide-resistance gene, in thisexample the BAR gene, which confers resistance to bialaphos. At 10- to14-day intervals, embryos were transferred to 563O medium. Activelygrowing putative transgenic embryogenic tissue were visible in 6-8weeks.

Example 15 Suppression of Maize PDS by a Modified Soybean ComplementaryRegion Regeneration of T₀ Plants

Transgenic embryogenic tissue is transferred to 288W medium [medium 288W contains the following ingredients: 950.000 ml of D-I H₂0; 4.300 g ofMS Salts; 0.100 g of Myo-Inositol; 5.000 ml of MS Vitamins StockSolution (No. 36J); 1.000 ml of Zeatin.5 mg/ml; 60.000 g of Sucrose;8.000 g of Agar (Sigma A-7049, Purified), which is added after Q.S. tovolume; 2.000 ml of IAA 0.5 mg/ml #; 1.000 ml of 0.1 Mm ABA #; 3.000 mlof Bialaphos 1 mg/ml #; and 2.000 ml of Agribio Carbenicillin 50 mg/ml#. Directions are: dissolve ingredients in polished deionized water insequence; adjust to pH 5.6; Q.S. to volume with polished deionized waterafter adjusting pH; sterilize and cool to 60° C. Add 3.5 g/L of Gelritefor cell biology. Ingredients designated with a # are added aftersterilizing and cooling to temperature] and incubated at 28° C. indarkness until somatic embryos matured, or about 10 to 18 days.Individual matured somatic embryos with well-defined scutellum andcoleoptile are transferred to 272 embryo germination medium [medium 272contains the following ingredients: 950.000 ml of deionized water; 4.300g of MS Salts; 0.100 g of Myo-Inositol; 5.000 of MS Vitamins StockSolution; 40.000 g of Sucrose; and 1.500 g of Gelrite, which is addedafter Q.S. to volume. Directions are: dissolve ingredients in polisheddeionized water in sequence; adjust to pH 5.6; Q.S. to volume withpolished deionized water after adjusting pH; and sterilize and cool to60° C.] and incubated at 28° C. in the light. After shoots and rootsemerge, individual plants are potted in soil and hardened-off usingtypical horticultural methods. Plants are then evaluated for thePDS-silenced phenotype.

Phytoene desaturase catalyzes a rate-limiting step in the biosynthesisof carotenoids in plants (Misawa, et al The Plant Journal (1993)4(5):833-840). It is a known target of bleaching herbicides such asnorflurazon. Cosuppression of the endogenous phytoene desaturase by theintroduced SHH3-flanked PDS 1 gives a similar bleached phenotype whenyoung plants are incubated in the light (Thomas, et al (2001) The PlantJournal 25(4):417-425; Kumagi et al (1995) PNAS USA 92:1679-1683; Ruizet al (1998) Plant Cell 10:937-946).

1-45. (canceled)
 46. A recombinant construct comprising SEQ ID NO:12 orSEQ ID NO:13.
 47. A vector comprising the recombinant construct of claim46.
 48. A cell comprising the recombinant construct of claim 60 whereinsaid cell is selected from the group consisting of a plant, an animal, aprotozoan, a bacterium, a virus and a fungus.
 49. A plant comprising inits genome the recombinant construct of claim
 60. 50. The plant of claim49 wherein said plant is a dicot.
 51. The plant of claim 50 wherein saidplant is a soybean plant.
 52. A seed obtained from the plant of claim49, 50 or 51 wherein said seed comprises the recombinant construct ofclaim 60.